Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos
整体原位杂交检测mRNA并提取DNA检测斑马鱼胚胎的基因分型    

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eLIFE
Apr 2018

 

Abstract

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to chromogenic or fluorescent visualization. Although recent fluorescent methods have allowed new capabilities such as single-molecule counting, qualitative chromogenic detection remains important for many applications because of its relative simplicity, low cost and high throughput, and ease of imaging using transmitted light microscopy. A remaining challenge is combining high contrast signals with reliable genotyping after hybridization. Dextran sulfate is commonly added to the hybridization buffer to shorten development times and improve contrast, but this reagent inhibits PCR-based genotyping. This paper describes a modified protocol for in situ hybridization in fixed whole mount zebrafish embryos using digoxigenin (DIG) labeled riboprobes that are detected with alkaline phosphatase conjugated anti-DIG antibodies and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates. To yield embryos compatible with downstream genotyping after hybridization without sacrificing contrast of the signal, this protocol omits dextran sulfate and utilizes a lower hybridization temperature.

Keywords: In situ, Hybridization, WISH, Zebrafish, Staining, Qualitative assay

Background

In situ hybridization is a technique that enables the detection of RNA in single cells, tissues or in whole embryos (Schulte-Merker et al., 1992; Hauptmann and Gerster, 1994; Schulte-Merker et al., 1994). The technique has been widely employed in developmental biology to characterize the spatial and temporal distributions of target mRNAs in fixed embryos (Thisse et al., 2001; Thisse and Thisse, 2004 and 2005). By allowing the comparison of mRNA distributions in wild-type, mutant and experimentally manipulated embryos, in situ hybridization continues to play a vital role in the investigation of gene function in development.

Hybridization is based on the principle of complementary base-pair binding of a specific probe oligonucleotide to a target mRNA. RNA oligonucleotides (riboprobes) are designed to pair with target mRNA molecules and are synthesized with nucleotides containing haptens, such as digoxigenin (DIG). In in situ hybridization, the riboprobes are incubated with previously fixed and permeabilized bulk tissue or intact embryos at an elevated temperature to favor the formation of hybrids with high sequence complementarity. The stable target-probe hybrids are then detected with a hapten-specific antibody. In chromogenic variations of the technique, the antibody is conjugated to alkaline phosphatase. This enzymatic activity results in the local generation of a colored precipitate upon the addition of a chromogenic substrate. NBT and BCIP are commonly used substrates yielding a purple-blue precipitate. Thus, the localization of target mRNA can be visualized with cellular resolution in the embryo or tissue by a colored signal in transmitted light microscopy.

The technique has been modified to compare the localization of two mRNAs by including different haptens on different probes (Hauptmann and Gerster, 1994) or combined with immunohistochemistry to compare the localization of mRNA and protein. Recent advances allow the fluorescent detection of multiple genes (Choi et al., 2010; Gross-Thebing et al., 2014), the sub-cellular localization of mRNAs (Jambor et al., 2015) and the determination of the number of transcripts in a sample (Wang et al., 2012; Little et al., 2013; Stapel et al., 2016; Trivedi et al., 2018). Nevertheless, qualitative chromogenic detection remains important in the field because of its relative simplicity, low cost, high throughput and ease of imaging.

The signal to noise ratio of the colored signal of an in situ hybridization experiment is a function of the specificity of riboprobe, i.e., the ability of the riboprobe to distinguish between different targets, and the stringency of the hybridization. High stringency requires the base pairing of all bases in the oligonucleotide while lower stringency allows some bases to be unpaired. The design of the riboprobe sets the specificity of hybridization. The reaction environment affects the stringency–annealing of oligonucleotides and the thermal stability of the resulting hybrid is influenced by temperature, pH, the concentration of monovalent cations and the presence of organic solvents in the environment.

Riboprobes have to be designed to be maximally complementary to the target mRNA for high specificity. Additionally, maximally complementary RNA-RNA hybrids have the highest thermal stability as base pair mismatches lower the thermal stability. The length of the riboprobe will affect the hybridization rate and also influence the thermal stability of the resulting hybrid–longer probes have a higher hybridization rate and generate thermally stable hybrids (Wetmur, 1976). Riboprobes of lengths between 300-3,200 base pairs specific to the target can be used with the protocol described here.

The temperature used for hybridization is determined from the melting temperature of the designed oligonucleotide. Hybridization is performed 15-25 °C lower than the calculated melting temperature (Wetmur and Davidson, 1968)–the higher this temperature, the greater the stringency of hybridization. However, prolonged incubation at typical RNA hybridization temperatures will degrade sample morphology. Therefore, organic solvents are added to reduce the thermal stability of the RNA duplexes, with formamide being the most common reagent (McConaughy et al., 1969). Hybridization buffers used in in situ hybridization also contain monovalent sodium cations, from sodium chloride and sodium citrate, which interact electrostatically with the RNA-RNA hybrid to stabilize it. Low salt washes are subsequently performed to reduce background by preferentially reducing the stability of non-specific hybrids.

Typical in situ protocols for the qualitative detection of mRNA in whole mount in zebrafish recommend a hybridization temperature of 70 °C (Thisse and Thisse, 2008) for high stringency. Such high stringency conditions are necessary if a riboprobe is required to discriminate between two similar target sequences. We found that for riboprobes with high specificity, by performing hybridization at lower temperatures (55-60 °C), we could achieve a more rapidly developing, higher contrast stain. We could further reduce the development time and enhance the contrast of the stain by increasing the effective concentration of riboprobe by adding dextran sulfate to the hybridization buffer (Lauter et al., 2011). Additionally, for probes that required prolonged development, the addition of polyvinyl alcohol to the NBT-BCIP staining solution reduces background (Kiyama and Emson, 1991). Making these modifications to the in situ hybridization protocol consistently accelerates the development and enhances the contrast of the stain.

A key part of an in situ experiment is the qualitative analysis of variations in mRNA distributions in mutant or otherwise experimentally manipulated embryos. However, differences in mRNA distributions may be evident before the genotype of the embryo can be ascertained by altered morphology. Further, such differences can be subtle, or variable, and may not fall into convenient Mendelian ratios complicating a straightforward assignment of wild-type and perturbed patterns. For these reasons, it is valuable to have a protocol compatible with reliable genotyping of embryos, or fragments of embryos, after hybridization and photographic documentation. We have observed that the presence of dextran sulfate in the hybridization buffer inhibits genotyping by PCR; therefore it is omitted if the embryos need to be genotyped after in situ hybridization.

The protocol described here describes three linked procedures: (1) the generation of DIG-labeled riboprobes suitable for hybridization; (2) in situ hybridization using a single riboprobe to qualitatively detect an mRNA target of interest in developing zebrafish embryos and imaging of the embryos; and (3) DNA extraction that gives a PCR-ready extract, enabling the presence or absence of indel mutations to be identified in the genomic DNA of individual, previously imaged embryos. We have successfully tested the protocol in labs in two different countries. Combined, these procedures form a protocol sufficiently detailed to enable a relatively inexperienced experimentalist to achieve high quality and reliable analysis of mRNA distribution patterns with a range of experimental perturbations.

Materials and Reagents

  1. Riboprobe synthesis
    1. 1.5 ml colorless microfuge tubes (Eppendorf, SafeLock, catalog number: 0030120086)
    2. PCR tubes (Sarstedt, Multiply®-μstrip Pro 8-strip, catalog number: 72.991.002)
    3. Restriction digest enzymes (Thermo Scientific, FastDigest) (store at -20 °C)
    4. 2x Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific, catalog number: F-565) (store at -20 °C)
    5. rNTP-DIG (Roche, catalog number: 11277073910) (store at -20 °C)
    6. RNase Inhibitor (RNaseOUT, Invitrogen, catalog number: 100000840) (store at -20 °C)
    7. RNA polymerases (1,000 U-20 U/μl) (store at -20 °C)
      1. Sp6 (Roche, catalog number: 10810274001)
      2. T3 (Roche, catalog number: 11031163001)
      3. T7 (Roche, catalog number: 10881767001)
    8. Clean up kits (store at room temperature)
      1. Qiaquick PCR purification kit (QIAGEN, catalog number: 28104)
      2. Promega SV Gel Wizard PCR purification kit (Promega, catalog number: A9281)
      3. RNeasy Min Elute (QIAGEN, catalog number: 74204)
    9. Agarose (Sigma-Aldrich, catalog number: A9539) (store at room temperature)
    10. 1x Tris-Acetate, EDTA (TAE) (store at room temperature, see Recipes)
      1. Tris Base (Sigma-Aldrich, BioXtra, catalog number: T6791) (store at room temperature)
      2. Glacial acetic acid (Suprapur®, EMD Millipore, catalog number: 1.00066) (store at room temperature)
      3. EDTA, 0.5 M (UltraPure, Invitrogen, catalog number: 15575-038) (store at room temperature)
    11. Hybridization buffer (Hyb) (store at -20 °C, see Recipes)
      1. Formamide, deionized (Roche, catalog number: 1814320) (store at 4 °C)
      2. 50 mg/ml Heparin stock (store at 4 °C, see Recipes)
        Heparin (Sigma-Aldrich, catalog number: H3400) (store at room temperature)
      3. Torula-RNA (Sigma-Aldrich, catalog number: R6875) (store at -20 °C)
      4. 20x SSC (store at room temperate, see Recipes)
        Sodium chloride (Sigma-Aldrich, BioXtra, catalog number: S7653)
        Trisodium citrate dihydrate (Sigma-Aldrich, catalog number: C8532) (store at room temperature)
      5. (Optional) Dextran sulfate sodium salt (Sigma-Aldrich, catalog number: D6001) (store at 4 °C)
      6. 10% Tween-20 (see Recipes)
        Tween-20 (Sigma-Aldrich, catalog number: P9416) (store at room temperature)

  2. Embryo culture
    1. 94 mm polystyrene Petri dish (Grenier Bio-One, catalog number: 633161)
    2. 100x PTU stock (store at room temperature, see Recipes)
      N-Phenylthiourea (PTU, Sigma-Aldrich, catalog number: P7629) (store at room temperature)

  3. In situ hybridization
    1. 24-well plates (Grenier Bio-One, Cell Star, catalog number: 662160)
    2. 2 ml colorless microfuge tubes (Eppendorf, SafeLock, catalog number: 0030120094)
    3. 3 ml plastic non-sterile, macrograduated Pasteur pipettes (HuberLab, catalog number: 15.4051.11)
    4. 94 mm polystyrene Petri dish (Grenier Bio-One, catalog number: 633161)
    5. Thin walled plastic container (Microwaveable plastic lunch box–any brand)
    6. Methanol (Fisher, catalog number: M/4000/15) (store at room temperature)
    7. Phosphate buffered saline, 0.1% Tween (PBST) (store at room temperature, see Recipes)
      1. Sodium chloride (Sigma-Aldrich, BioXtra, catalog number: S7653) (store at room temperature)
      2. Potassium chloride (Sigma-Aldrich, BioXtra, catalog number: P9333) (store at room temperature)
      3. Sodium phosphate dibasic (Sigma-Aldrich, BioXtra, catalog number: S7907) (store at room temperature)
      4. Potassium phosphate monobasic (Sigma-Aldrich, catalog number: P9791) (store at room temperature)
      5. 10% Tween-20 (see Recipes)
    8. 4% Paraformaldehyde (PFA) in PBST (store at 4 °C) (see Recipes)
      1. PFA powder (Sigma-Aldrich, catalog number: P6148) or 16% PFA (Alfa Aesar, catalog number: 43368.9M) (store at 4 °C)
      2. PBST (see Recipes)
    9. Proteinase K stock (store at -20 °C, see Recipes)
      Proteinase K (Merck, catalog number: 1.24568) (store at 4 °C)
    10. Hybridization buffer (Hyb) (store at -20 °C, see Recipes)
    11. Post-hybridization washes (see Recipes)
      1. Wash 1
      2. Wash 2
      3. Wash 3

  4. Detection
    1. AP-conjugated anti-DIG antibody (Anti-Digoxigenin-AP, Fab fragments from sheep) (Roche, catalog number: 11093274910) (store at 4 °C)
    2. Maleic acid buffer, 0.1% Tween (MABT) (store at room temperature, see Recipes)
      1. Maleic acid (Sigma-Aldrich, catalog number: M0375) (store at room temperature)
      2. Sodium chloride (Sigma-Aldrich, BioXtra, catalog number: S7653) (store at room temperature)
      3. Sodium hydroxide (Fisher Chemical, catalog number: S/4920/60) (store at room temperature)
      4. 10% Tween-20 (see Recipes)
    3. 10% Blocking reagent in maleic acid buffer 
      1. Blocking Reagent (Roche, catalog number: 11096176001) (store at room temperature)
      2. Maleic acid buffer (see Recipes)
    4. 2% Blocking reagent in MABT (2% Roche block) (see Recipes)
    5. Staining buffer (see Recipes)
      1. Tris-HCl (Sigma-Aldrich, BioXtra, catalog number: T6666) (store at room temperature)
      2. Magnesium chloride hexahydrate (Sigma-Aldrich, BioXtra, catalog number: M2670) (store at room temperature)
      3. Sodium chloride (Sigma-Aldrich, BioXtra, catalog number: S7653) (store at room temperature)
    6. Staining solution (see Recipes)
      1. NBT (Roche catalog number: 11383213001) (aliquot and store at -20 °C)
      2. BCIP (Roche, catalog number: 11282221001) (aliquot and store at -20 °C)
      3. Staining buffer (see Recipes)
      4. (Optional) Polyvinyl alcohol (PVA, Sigma-Aldrich, catalog number: P1763) (store at room temperature)

  5. Mounting
    1. 60 mm Petri dish
    2. A human eyelash glued to a tooth pick/syringe needle or a fine pipette tip (GELoader Tips 0.5-20 μl, 62 mm, Eppendorf, catalog number: 0030001222)
    3. (Optional) Mold for embryos for photographic documentation in whole mount [the mold negative is made in our laboratory (Herrgen et al., 2009)]
    Materials for flat mounting:
    1. (Optional) Dissection hook (Picker et al., 2009) made with Tungsten wire (Hamilton, catalog number: 18306)
    2. 10 μl pipette tip 
    3. Glass slides (Thermo Scientific, Superfrost, catalog number: 12372098)
    4. Glass coverslip (VWR, Menzel Gläser, #1.5 rectangular 22 x 40 mm, catalog number: 631-1370)
    5. Silicone grease or petroleum jelly (such as Vaseline)
    6. Transparent nail varnish (any brand)
    7. 87% glycerol in distilled water (store at room temperature, see Recipes)
      Glycerol (Fisher, catalog number: G/0650/08) (store at room temperature)

  6. Genomic DNA extraction (store at room temperature)
    1. 50x base solution (see Recipes)
      1. Potassium hydroxide (Fisher Chemical, catalog number: P/5640/60)
      2. EDTA, 0.5 M (UltraPure, Invitrogen, catalog number: 15575-038)
    2. 1x base solution (see Recipes)
    3. 50x neutralization solution (see Recipes)
      Tris-HCl (Sigma-Aldrich, catalog number: T6666)
    4. 1x neutralization solution (see Recipes)

Equipment

  1. Stock solution preparation
    1. Magnetic stirrer (Heidolph Instruments, model: MR Hei-Standard, catalog number: 505-20000-00)
    2. pH meter (Mettler Toledo, model: Seven compact and InLab Expert Pro-ISM)

  2. Riboprobe synthesis
    1. PCR machine (Eppendorf, model: Mastercycler pro S) 
    2. Thermoblock (Eppendorf, model: ThermoMixer C and SmartBlock 2.0 ml)
    3. Gel electrophoresis (Bio-Rad, model: PowerPac Basic Power Supply; Wide Mini-Sub Cell GT Cell)
    4. NanoDrop spectrophotometer (Thermo Fisher, model: NanoDropTM 2000, catalog number: ND 2000)

  3. In situ hybridization
    1. Forceps (Dumont, model: 5-Inox-E)
    2. Stereomicroscope (Olympus, model: SZ61)
    3. Shaker (Heidolph, model: Duomax1030, catalog number: 543-32205-00)
    4. (Optional) Vacuum set-up (Integra, Vacusafe aspiration system)
    5. Water bath (GFL, model: 1003)
    6. (Optional) Well plate stand (Diversified Biotech, catalog number: WPST-1000)

  4. Documentation
    1. RGB Camera (Olympus, model: DP22)
    2. Ring light (Photonic Optics, model: LED 40 ring light)

  5. Genomic DNA extraction
    1. Micro knife (Fine Science Tools, catalog number: 10315-12)

Software

  1. Fiji (Schindelin et al., 2012, https://fiji.sc/)
  2. Snapgene (GSL Biotech LLC)

Procedure

  1. Riboprobe synthesis
    Note: DNA templates for riboprobes can be either cDNA cloned into a plasmid backbone downstream of an RNA polymerase promoter or PCR product amplified from cDNA or genomic DNA. See Note 1.
    1. Template preparation
      Preparing a template from a plasmid
      1. Linearize the plasmid by digestion with the appropriate restriction enzyme at 37 °C. Choose an enzyme such that the template is linearized immediately downstream of the cDNA insert.
        Set the reaction up as:
        Plasmid template
        5 μg
        Restriction enzyme
        5 μl
        10x restriction buffer
        10 μl
        Make up to 100 μl with distilled water/nuclease-free water.
      2. Purify the linearized plasmid with a commercial clean up kit. We recommend the Qiaquick PCR purification kit. Purify the plasmid as per the manufacturer’s instructions and elute the plasmid in 30 μl of elution buffer.
      3. Dilute 1 μl of the eluent in 4 μl of distilled water. Use 1 μl to assess concentration on a spectrophotometer and 4 μl to check the linearization with agarose gel electrophoresis. A single band corresponding to the linearized plasmid should be visible on the gel. Proceed with riboprobe synthesis if a single band corresponding to the fully linearized plasmid is visualized. 

      Preparing a template by PCR
      1. Design primers to amplify the desired target from cDNA or genomic DNA. To make a riboprobe that is complementary to the target mRNA, add the RNA polymerase (T7/T3/Sp6) promoter sequence to the reverse primer as: 5’-RNA polymerase promoter sequence + template specific sequence-3’.


      2. Carry out the PCR with the optimal cycling conditions. We recommend using a high fidelity DNA polymerase like Phusion.
      3. Clean up the PCR product. We recommend the Promega SV Gel Wizard PCR purification kit. Elute in 30 μl of the kit elution buffer. Dilute 1 μl of the eluent in 4 μl of distilled water. Use 1 μl of this dilution to assess concentration on a NanoDrop and 4 μl to assess product quality by gel electrophoresis. Proceed with riboprobe synthesis if a single band of the appropriate length is obtained. It is not necessary to sequence the PCR product to confirm sequence accuracy of the template (see Note 6).
    2. In vitro transcription
      1. Thaw the reaction components (except the enzymes) and place on ice.
      2. Set up each 20 μl transcription reaction as:
        DNA Template
        1-1.5 μg of linearized plasmid
        or 100-500 ng of PCR product
        10x Transcription buffer
        2 μl
        rNTP-DIG
        2 μl
        RNase Inhibitor
        0.5 μl
        RNA polymerase
        2 μl
        Adjust the volume to 20 μl with RNase-free water, if needed.
      3. Transcribe for 2-4 h at 37 °C. The yield of the reaction is equivalent when the transcription time is between 2 h and 4 h.
      4. Purify the riboprobe from the reaction with a commercial kit like QIAGEN RNeasy Min Elute. Follow the manufacturer’s instructions and elute the riboprobe in 30 μl of RNase-free water. Dilute 1 μl of the probe in 4 μl of water. Use 1 μl to assess the concentration on a spectrophotometer and 4 μl to assess the quality of the transcription by agarose gel electrophoresis. The typical A280/A260 ratio of the synthesized probe after clean up is between 1.85 and 2.00. To resolve the RNA riboprobe on a 1% agarose gel in 1x TAE, run it at 140 V for 10-15 min. The riboprobe should resolve as a distinct band. Occasionally, a longer band is also obtained–this is typically an electrophoresis artifact arising due to RNA secondary structure, if a high-quality PCR product or completely linearized plasmid template has been used.
      5. Store the remainder of the synthesized riboprobe at -20 °C. Dilute the riboprobe in 5 ml of Hyb to perform in situ hybridization. See Note 2.
      6. Test the probe by performing in situ hybridization and assessing the pattern of hybridization obtained. 

  2. In situ hybridization
    Notes: 
    1. This protocol has been optimized for embryos up to 48 h post fertilization (hpf) and is carried out in 24-well plates. Use 800 μl for washes and 500 μl for incubations. When exchanging media, the embryos being stained should not be allowed to dry out and should always remain covered with liquid. To ensure that they do not get stuck to the bottom or on to the sides of the wells, pipette gently and check after each solution change that embryos are covered by liquid.
    2. All incubation steps, unless specified, are performed at room temperature on a shaker. 

    1. Fixation and dehydration of embryos (see Note 3).
      Fixing embryos younger than 24 hpf
      1. Transfer embryos into 2 ml microfuge tubes (for long-term storage) or into wells of a 24-well plate (for immediate use for in situ) with a 3 ml plastic Pasteur pipette. Up to 100 embryos can be stored in a single tube and up to 30 embryos can be processed per well in a 24-well plate.
      2. Remove as much embryo medium as possible. If the embryos are in their chorions, all the liquid can be removed. Add 4% PFA in PBST (fix)–1 ml per tube, 800 μl per well. Incubate on a shaker for 5 min at room temperature. This step serves to remove any excess embryo medium.
      3. Exchange the fix with fresh fix–up to 2 ml per tube, 800 μl-1 ml per well. Incubate at room temperature for 1 to 4 h or overnight at 4 °C, with shaking.
      4. Remove the fix and add PBST–up to 2 ml per tube, 800 μl-1 ml per well.
      5. Transfer embryos into PBST in a 35 mm Petri dish with a 3 ml plastic Pasteur pipette and use forceps to dechorionate the embryos under a stereo microscope (Video 1). Transfer embryos back into the well plate or into a fresh tube/vial. The pipette should be rinsed by pipetting up and down in PBST, and the PBST should be exchanged between batches of embryos to prevent cross-contamination. 

      Fixing embryos older than 24 hpf
      1. From 24 hpf onwards, add PTU to the embryo medium to a final concentration of 0.003% to inhibit pigment development in the embryos.
      2. Dechorionate embryos in embryo medium in a 35 mm Petri dish under a stereomicroscope (Video 2) and transfer them with a 3 ml plastic Pasteur pipette to the well plate or microfuge tube vial–30 for a well, 100 for a tube. The embryo medium should be exchanged between batches of embryos to prevent cross-contamination.
      3. Remove as much embryo medium as possible. Add 4% PFA in PBST (fix)–1 ml per tube, 800 μl per well. Incubate on a shaker for 5 min at room temperature. This step serves to remove any excess embryo medium.
      4. Exchange with fresh fix–up to 2 ml per tube or vial, 800 μl to 1 ml per well in the well plate. Incubate with shaking at room temperature for 1 to 4 h or overnight at 4 °C.
      5. Briefly rinse the embryos by removing fix and adding PBST–up to 2 ml per tube, 800 μl-1 ml per well.
      6. Exchange the PBST and wash the embryos in PBST 3 times, for a duration of 5 min each time, on a shaker at room temperature.

      Dehydration and permeabilization of embryos
      1. Dehydrate the embryos in a graded methanol series: add 800 μl of 30% methanol in PBST, then replace with 800 μl of 50% methanol in PBST and then with 800 μl of 70% methanol in PBST. Incubate on a shaker between each replacement for 5 min.
      2. Incubate the embryos in 800 μl of absolute methanol for 5 min on a shaker.
      3. Replace the methanol and incubate embryos at -20 °C overnight to permeabilize the embryos. Use 1.1 ml of methanol per well or 2 ml per microfuge tube.
        The protocol can be stopped here if necessary as embryos can be stored at -20 °C in methanol indefinitely (see Note 3).

        Video 1. Dechorionation of fixed somitogenesis stage embryos


        Video 2. Dechorionation of live 36 hours post fertilization (hpf) embryos

    2. Enzymatic embryo permeabilization
      1. Rehydrate embryos in a graded methanol series: add 800 μl of 70% methanol in PBST, then replace with 800 μl of 50% methanol in PBST and then with 800 μl of 30% methanol in PBST. Incubate on a shaker for 5 min at each step. 
      2. Perform two 5-min washes with PBST.
      3. Digest embryos in Proteinase K (PKA). Dilute the PKA stock 1:200 for embryos younger than 24 hpf and 1:100 for embryos older than 24 hpf in PBST. Incubate in 500 μl without shaking at room temperature. Digestion time is embryonic stage dependent; the timings listed below can be used as a guideline. Stages indicated here are as per Kimmel et al. (1995).



      4. Stop the reaction by making a rapid exchange with PBST followed by a 20-min re-fixation with 500 μl of fix at room temperature on a shaker.
      5. Remove the fix; perform one rapid wash with PBST followed by two 5-min PBST washes.
    3. Hybridization
      Note: The temperature used for hybridization is probe-dependent, but a good starting temperature is 60 °C (see Note 4). Perform hybridization steps in a water bath set to the hybridization temperature. Use a thin-walled plastic container lined with moistened tissue to incubate the well plate in the water bath. The container and hybridization solutions need to be pre-warmed to the hybridization temperature in the water bath before use.
      1. Pre-hybridize in 500 μl of Hyb at the hybridization temperature for a minimum of 30 min. Embryos can be pre-hybridized for up to 3 h at the hybridization temperature. 
      2. Remove the Hyb and replace it with 500 μl of labeled riboprobe diluted in Hyb. Hybridize overnight at the hybridization temperature. See Note 5.
    4. Post-hybridization washes
      Note: These washes are also performed in a water bath at the hybridization temperature. Prepare the wash solutions fresh and warm them in the water bath before use. Timings indicated in this step are critical–do not wash for shorter or longer than the time recommended.
      1. Remove and recycle probe. Store it at -20 °C. See Note 2.
      2. Wash embryos 2 times, for 30 min each time in Wash 1.
      3. Wash embryos for 15 min in Wash 2.
      4. Wash embryos 2 times, for 30 min each time in Wash 3.
    5. Detection
      1. Make a quick rinse with MABT to remove any remaining Wash 3 from the embryos.
      2. Add 500 μl of 2% blocking reagent and incubate without shaking in the dark at room temperature for at least 30 min.
      3. Dilute AP-conjugated anti-DIG antibody 1:2,000 in 2% blocking reagent. Remove the blocking reagent and add 500 μl of the diluted antibody in blocking reagent to the embryos. Incubate for 2 h, without shaking, at room temperature in the dark.
      4. Remove antibody solution and make 2 quick washes with MABT.
      5. Wash in 10-20 min intervals over 1 to 2 h at room temperature; at least 6 exchanges of MABT should be made. Alternatively, embryos can be washed at 4 °C overnight in MABT, followed by three 5-min washes the next day.
      6. Equilibrate embryos in freshly prepared staining buffer twice for 5 min each.
      7. Add 500 μl of the staining solution to the embryos. Stain in the dark at room temperature without shaking. See Note 5.
      8. Control the staining reaction by eye under a stereomicroscope. Place the well plate on a white background and illuminate the plate from above. Monitor the development of the embryos in the first 5 min after the staining solution has been added and then in 10-15 min intervals till the stain saturates or the desired contrast is achieved.
      9. Stop the staining reaction by performing three 5-min washes in PBST.
      10. Incubate embryos in methanol at room temperature till the yolk is cleared and appears white, or overnight at 4 °C or -20 °C.
        Note: See Figures 2, 3B and 4B for stains we have made with this protocol. See Note 6 for notes on troubleshooting.
    6. Mounting and photography
      1. Remove the methanol from the embryos by making two 5-min washes in PBST.
      2. Re-fix in 4% PFA in PBST for at least 15 min at room temperature.
      3. Wash embryos twice each for 5 min in PBST.
      4. Add 87% glycerol to the embryos and leave to equilibrate at 4 °C. When the embryos sink to the bottom of the well, they are completely equilibrated. This takes ~12 h. Embryos can be subsequently stored in 87% glycerol either at 4 °C or -20 °C.
      5. Photograph the embryos: 
        1. Photographing whole mount preparations:
          Photograph the embryos in 87% glycerol in a 35 mm Petri dish or an imaging mold (see Note 7) with an RGB camera coupled to a stereomicroscope. Illuminate the embryos from above using a ring light. Place a white sheet of paper below the dish to provide a white background for the image. An eyelash, tungsten wire or fine pipette tip can be used to manipulate the embryos. 
        2. Photographing flat mount preparations:
          See also the JoVE video protocol for de-yolking and flat mounting (Cheng et al., 2014).
          1)
          Transfer the embryos to be flat mounted onto a glass slide with a drop of 87% glycerol. De-yolk the embryos with an eyelash or sharpened tungsten wire (see Note 8) in the drop. Move the embryo to the edge of the drop so that a minimal volume of glycerol covers it. Remove the remaining yolk cells with the eyelash. Periodically rinse the loosened yolk cells off the embryo in the glycerol drop. Move the embryo to a fresh drop of glycerol after de-yolking.
          2)
          Place the embryonic axis, dorsal side up, on a clean glass slide with the wire or the eyelash. Place approximately 15-20 μl total volume of 87% glycerol in small drops next to the embryos. Up to 10 de-yolked embryos can be flat mounted on a single slide.
          3)
          Make 4 small grease pillars with a 10 μl pipette tip to hold the corners of a glass coverslip in place. Carefully lower the coverslip onto the slide (Figure 1).
          4)
          Seal the 4 edges of the coverslip with transparent nail varnish.
          5)
          Photograph the embryos with transmitted light on a stereoscope with an RGB camera.


          Figure 1. Preparing a flat mount. A. Placing drops of glycerol next to de-yolked embryos (circled) and gently lowering a coverslip on the slide that will be held in place by grease pillars at the corners. B. The finished slide with flat mounted embryos (circled) and coverslip edges sealed with transparent nail varnish.

  3. Genotyping
    Note: Genomic DNA can be extracted from intact embryos or from tissue fragments after in situ hybridization. Fragments of tissue are taken before de-yolking and flat mounting the embryos. Perform the following steps in PCR tubes–1 embryo per tube.
    1. Select the embryos to be genotyped and rinse them together in a small Petri dish or singly in PCR tubes with PBST to remove the glycerol. A few rapid exchanges followed by three 5-min washes on a shaker at room temperature are typically sufficient.
    2. Transfer the whole embryo with forceps into a PCR tube. If DNA is being extracted from tissue fragments, transfer the embryo onto a glass slide into a 15-20 μl drop of PBST. Cut one-third to one-half of the embryo that is not stained with a micro knife. Transfer the piece to a PCR tube. Ensure that no excess liquid is transferred. Add 87% glycerol to the tissue on the glass slide to continue with de-yolking and flat mounting. 
    3. Add 15 μl of 1x base solution to the tube and incubate at 95 °C for 30 min.
    4. Bring to room temperature and add 15 μl of 1x neutralization solution.
      Note: This solution is PCR ready (see Note 9).

Data analysis

  1. Whole mount mRNA in situ hybridization coupled to chromogenic detection is a qualitative assay for gene expression in the embryo. In the whole mount format, the distribution of the mRNA of interest in specific tissue(s) at a developmental stage of interest can be assessed. Performing mRNA in situ hybridization for a target in embryos fixed at different developmental stages can help the investigator characterize temporal changes in the mRNA expression of the gene of interest over the course of development.
    In situ hybridization can also be utilized to assess the effect of the loss or knock down of gene function on the expression of genes of interest. For example, in situ hybridization has been used in our laboratory to assay the wave patterns of cyclic genes arising from their transcriptional oscillations in zebrafish embryos during somitogenesis and to demonstrate that loss of function mutant cyclic gene alleles lead to the loss of these wave patterns (Figure 1, Lleras Forero et al., 2018). Working from a set of microscope images of embryos, in situ hybridization can also be used as a semi-quantitative endpoint assay to measure the spatio-temporal effects of an experimental perturbation, for example to investigate the upregulation or downregulation of Wnt signaling (Figures 3, 5 and 6, Bajard et al., 2014). mRNA in situ hybridization has also been used to perform a comparison between the spatial distribution of endogenous cyclic gene expression and a transgenic reporter (Figure S1, Soroldoni et al., 2014). However, care must be taken in interpreting results that depend on the intensity of the chromogenic signal, either qualitatively, or after quantifying the intensity of the signal with a tool such as plot profile in Fiji (https://fiji.sc/), as the intensity of the stain is initially development time-dependent, but the enzymatic reaction can later saturate.
  2. Designing a genotyping strategy from the extracted genomic DNA will depend entirely on the nature of the specific alleles in the experiment. For some alleles, base transitions will lead to the gain or loss of a site for restriction digestion, allowing for a restriction digestion-based analysis of the PCR amplicon obtained from the genomic DNA using primers specific for the gene of interest. When such a strategy is not possible, the PCR amplicon can be sequenced. Analysis of genotyping sequence data generated can be done with open source or commercially available software (Snapgene, GSL Biotech LLC). For example, we detected base-pair transitions causative for a TALEN induced her1 mutation in a her7hu2124 background initially by sequencing (Figure S1, Lleras Forero et al., 2018). The mutation led to the loss of a restriction site and embryos were subsequently genotyped by the resistance of the her1 mutant allele to restriction digestion.

Notes

  1. Riboprobe design
    In general, using long probes provides a good stain. Long probes yield hybrids with high thermal stability and provide greater signal amplification. Therefore, a good general approach is to design a riboprobe that is complementary to the entire coding sequence of the mRNA. However, such an approach might not be suitable in all cases. For example, to distinguish between mRNAs of paralogs, designing a shorter riboprobe with high complementarity to the paralog of interest is the better approach. In our hands, probes of approximately 300-3,200 bp lengths have reliably yielded a good stain with this protocol (Figure 2). 


    Figure 2. Representative results of the in situ hybridization protocol using riboprobes of lengths ~300 and 3,200 base pairs. A. Stain for paraxial protocadherin (papc/pcdh8) using a riboprobe of 3,137 base pair length, antisense to the coding region of papc mRNA (Yamamoto et al., 1998). B. Stain for ripply1 using a 325 base pair riboprobe, antisense to the coding region of ripply1 mRNA (Lleras et al., 2018). Hybridization has been performed at 60 °C on 10-somite stage wild-type embryos. Flat mounted preparations are shown here oriented with the posterior towards the bottom of the panels. Scale bar is 50 μm and applies to both panels. 

  2. Probe concentration
    The final working concentration of the probe should be optimized experimentally. In general, concentrations of 0.3-1 ng/μl produce a good in situ stain; however, working concentrations should be titrated every time a new probe is synthesized. The probe can be recycled and reused many times. Note down the initial volume, as the probe will dilute over time and fail to produce a high contrast stain.
  3. Embryo fixation and storage
    Embryos can be fixed and dehydrated ahead of time. The quality of the fix will affect the signal to noise ratio of the stain. Freshly prepared fix gives the best results, but fix up to a week old can be used. Embryos should not be incubated in fix for more than 4 h at room temperature or 1 week at 4 °C. Fixed and dehydrated embryos can be stored in methanol at -20 °C indefinitely.
  4. Hybridization temperature
    The hybridization temperature is probe-dependent and has to be experimentally determined for each probe. The choice of temperature also sets the stringency of the protocol–stringency can be increased by increasing the hybridization temperature and decreased by lowering it. When using probes that need to discriminate between targets with high sequence similarity, the stringency of the protocol needs to be raised to favor the formation of the desired RNA hybrids as a 1% base pair mismatch decreases the thermal stability of RNA-RNA duplexes by roughly 1 °C (Wetmur et al., 1976).
    In our hands, a good starting temperature has been 60 °C for probes designed to detect the coding sequence of the target mRNA (Figures 2 and 3). For short probes and intronic probes, 55 °C has been used as the starting temperature. The hybridization temperature has then been optimized by changing the temperature in 2 °C steps.
     

    Figure 3. Hybridization at 60 °C is sufficiently stringent to achieve a high contrast final stain with a specific probe. In situ hybridization to detect xirp2a (Deniziak et al., 2007) performed at 70 °C and 60 °C in 36 hpf wild-type embryos. A. Stain after 110 min of development when probe is hybridized at 70 °C. B. Stain after 50 min of development when hybridized at 60 °C. Riboprobe concentration used is 0.5 ng/μl in hybridization buffer. Scale bar is 100 μm and applies to both panels.

  5. Improving contrast of the stain
    Adding dextran sulfate to the hybridization buffer will yield high contrast stains with a relatively short development time, compared to stains produced without dextran sulfate (Figure 4). If long development times are required to detect the target mRNA, adding 10% PVA to the staining solution will decrease the rate at which background staining will develop.


    Figure 4. Addition of dextran sulfate to the hybridization buffer enhances the contrast of the final stain. In situ hybridization to detect xirp2a performed at 60 °C in 36 hpf wild-type embryos. A. Stain after 25 min of development when probe is hybridized in hybridization buffer B. Stain after 25 mins of development when probe is hybridized in hybridization buffer with 5% dextran sulfate (DS). Riboprobe concentration used is 1 ng/μl. Scale bar is 100 μm and applies to both panels.

  6. Troubleshooting
    No signal from an antisense riboprobe
    In vitro transcription has not been successful: The integrity of the plasmid could have been compromised. Re-prep the plasmid or generate a template by PCR.

    Sub-optimal signal to noise ratio
    1. Poor quality probe: Control for probe quality by performing agarose gel electrophoresis of templates and synthesized riboprobes. They should resolve as distinct bands. Templates can be sequenced to assess sequence accuracy.
    2. Probe concentration is too high: Diluting the probe can reduce the background.
    3. Background due to non-specific hybridization: Increase the stringency of the protocol by raising the hybridization temperature.
    4. Background due to prolonged development: Reduce the hybridization temperature to lower the stringency of the protocol, add 10% PVA to the staining solution.

    Poor signal intensity
    1. Probe is too dilute: Synthesize new probe, increase the concentration of the probe.
    2. Probe design: Increase the length of the probe for better signal amplification.
    3. Hybridization is too stringent: Decrease the hybridization temperature.
    4. Add 5% dextran sulfate to the hybridization buffer. 
  7. Mold for photography of whole mount preparations
    An imaging dish for photographing whole mount specimens can be made by casting a mold for the embryos in 1-1.5% agarose in distilled water using a polydimethylsiloxane (PDMS) negative. This negative is cast from a positive made from Perspex plastic machined with the conical depressions of the desired diameter and depth (Figure 4B, the positive and negative for the imaging mold are made by our laboratory and are described in Herrgen et al., 2009). Add 87% glycerol to the dish. Transfer the stained embryos into the mold and orient them using the tip of a tungsten wire, an eyelash or a fine pipette tip such that their yolks fit in the conical depressions under a stereomicroscope (Figure 5).


    Figure 5. Preparing to document the whole mount in situ hybridization preparations. A PDMS negative with an array of raised cones of 0.8 mm diameter and 0.4 mm height is used to cast the agarose mold with conical depressions. The yolk of the embryos fits into the depressions and embryos can be oriented laterally such that they lie flat on the surface of the agarose. A. Casting a mold to orient embryos in 1%-1.5% agarose. B. Embryos are oriented in the conical cavities of the mold in 87% glycerol. C-C’. Polydimethylsiloxane (PDMS) negative used to cast the mold. D. Agarose mold for the embryos. E. Embryos oriented for photography. 

  8. De-yolking the embryo
    1. We recommend referring to the video protocol for de-yolking and flat mounting (Cheng et al., 2014).
    2. A sharpened tungsten wire or “hook” can be used instead of an eyelash for de-yolking embryos. To sharpen and shape the wire, fire-polish the end of the tungsten wire by holding the last 0.5 to 1 cm length of the wire in a Bunsen burner flame until it glows orange for 30-60 s. With a pair of forceps shape the flamed wire into a hook in 87% glycerol. Detailed instructions to make the tungsten hook and holder are provided in Picker et al., 2009.
  9. Using the embryo extract for PCR
    A dilution series ranging from no dilution up to 1:5 in distilled water should be trialed to find the optimal volume of genomic DNA extract that is necessary for PCR for a given embryonic stage. Extracts from 8 to 10-somite stage whole embryos and embryo fragments typically do not need any further dilution and 1-2 μl can be used per 10 μl PCR reaction. Extracts from tissue of 24-36 hpf embryos typically need to be diluted 1:4.

Recipes

Note: This protocol does not require DEPC treated water or solutions or specific RNase free conditions. Distilled or ultrapure water, autoclaved glassware and single use polypropylene consumables such as microfuge tubes, centrifuge tubes, plastic Pasteur pipettes micropipette tips are sufficient.

  1. 1x TAE
    40 mM Tris-acetate
    20 mM Acetate
    1 mM EDTA
    pH 8.3
    Dilute 10x TAE 1:10 in ultrapure water
    Store at room temperature
    To make 1 L of 10x TAE (0.4 M Tris-acetate, 0.2 M Acetate and 0.1 M EDTA):
    48.5 g Tris base
    11.4 ml glacial acetic acid
    20 ml 0.5 M EDTA, pH 8.0
    Make up to 1 L with ultrapure water
    Autoclave
  2. 100x PTU stock (0.3% [weight by volume] in distilled water) (100 ml)
    Dissolve 0.3 g N-Phenylthiourea (PTU) in 100 ml of distilled water with heating and stirring (store in the dark at room temperature)
  3. 10% Tween-20 (dilute Tween-20 1:10 in distilled water) (100 ml)
    10 ml Tween-20
    Make up to 100 ml with distilled water
    Stir till a homogeneous solution is achieved
    Store at room temperature
  4. PBST
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4 in distilled water, pH 7.4
    0.1% Tween-20
    To make 1 L PBST:
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Make up to 1 L with distilled water after adjusting pH and autoclave
    Add 10 ml of 10% Tween-20
    Store at room temperature
  5. 4% PFA in PBST (4% weight by volume in PBST) (100 ml)
    1. Pre-warm 100 ml PBS to 68 °C in a water bath
      Add 4 g PFA
      Incubate at 68 °C with periodic agitation for 50 – 60 mins
      Add 1 ml of 10% Tween-20 when the solution has cooled to room temperature
      Store for up to a week at 4 °C
      Note: The PFA should dissolve in 50 min to 1 h
      OR
    2. Dilute 16% PFA 1:4 in PBST. Store for up to a week at 4 °C
  6. Proteinase K stock
    1 mg/ml in distilled water
    Store as aliquots at -20 °C
  7. 20x SSC
    3 M NaCl
    300 mM tri-sodium citrate
    pH 7.0-7.2
    To make 1 L 20x SSC:
    175.2 g NaCl
    88.2 g tri-sodium citrate
    Make up to 1 L with distilled water after adjusting pH
    Autoclave
    Store at room temperature
  8. 50 mg/ml Heparin stock
    Dissolve in Heparin in distilled water to a final concentration of 50 mg/ml
    Store at 4 °C
  9. Hybridization buffer (Hyb)
    50% Formamide
    5x SSC
    50 μg/ml Heparin
    0.5 mg/ml Torula-RNA
    0.1% Tween-20
    Optional: 5% weight by volume dextran sulfate
    To make 50 ml Hyb:
    25 ml formamide
    12.5 ml 20x SSC
    50 μl Heparin stock
    25 mg Torula-RNA
    2.5 g dextran sulfate
    Vortex well till a homogenous is achieved
    Add 500 μl 10% Tween-20
    Make up to 50 ml with distilled water
    Store at -20 °C
    Note: Dextran sulfate is omitted from hyb used for pre-hybridization.
  10. Post hybridization washes
    Wash 1: 50% Formamide, 2x SSC, 0.1% Tween-20 in distilled water
    To make 50 ml:
    25 ml Formamide
    5 ml 20x SSC
    500 μl 10% Tween-20
    Make up to 50 ml with distilled water
    Wash 2: 2x SSC, 0.1% Tween-20 in distilled water
    To make 50 ml:
    5 ml 20x SSC
    500 μl 10% Tween-20
    Make up to 50 ml with distilled water
    Wash 3: 0.2x SSC, 0.1% Tween-20 in distilled water
    To make 50 ml:
    500 μl 20x SSC
    500 μl 10% Tween-20
    Make up to 50 ml with distilled water
    Note: Make the appropriate volume of wash solution fresh.
  11. MABT
    100 mM Maleic Acid
    150 mM NaCl in distilled water
    pH 7.5
    0.1% Tween-20
    To make 1 L:
    8.77 g maleic acid
    11.6 g NaCl
    Adjust pH with NaOH pellets
    Make up to 1 L with distilled water
    Autoclave
    Add 10 ml 10% Tween-20
    Store at room temperature
  12. 10% blocking reagent (Roche block)
    Prepare 10% blocking reagent in MAB as per the manufacturer’s instructions
    Store at -20 °C as aliquots
  13. 2% Blocking reagent in MABT (2% Roche block)
    Dilute 10% blocking reagent (Roche block) 1:5 in MABT
    Make fresh
  14. Staining buffer
    100 mM Tris-HCl, pH 9.5
    100 mM NaCl
    50 mM MgCl2 in distilled water
    To make 50 ml:
    5 ml 1 M Tris-HCl, pH 9.5
    5 ml 1 M NaCl
    2.5 ml 1 M MgCl2
    500 μl 10% Tween-20
    Note: Make fresh.
  15. Staining solution
    4.5 μl of NBT and 3.5 μl of BCIP per 1 ml of the staining buffer
    Vortex to mix
    Protect from light
    Note: Prepare the appropriate volume fresh.
    Optional: add PVA to a final concentration of 10%, weight by volume
    To make 10 ml of staining solution with 10% PVA:
    Add 1 g PVA to 10 ml of staining buffer (without Tween-20)
    Bring into solution with stirring and heating
    Cool to room temperature
    Add 45 μl NBT and 35 μl of BCIP
    Vortex to mix
    Protect from light
    Note: 10 ml is the minimum volume that can be made.
  16. 87% glycerol in distilled water (weight by weight)
    To make 100 g:
    87 g glycerol in 13 g distilled water
    Stir till a homogenous solution is achieved
    Store at room temperature
  17. 50x base solution
    1.25 M KOH
    10 mM EDTA in distilled water
    To make 200 ml:
    14 g KOH
    4 ml 0.5 M EDTA
    Add distilled water to a final volume of 200 ml
    Store at room temperature
  18. 1x base solution
    Dilute 50x base solution 1:50 in distilled water
    Note: Prepare the appropriate volume fresh.
  19. 50x neutralization solution
    2 M Tris-HCl in distilled water
    To make 200 ml:
    63 g Tris-HCl
    Add distilled water to a final volume of 200 ml
    Store at room temperature
  20. 1x neutralization solution
    Dilute 50x neutralization solution 1:50 in distilled water
    Note: Prepare the appropriate volume fresh.

Acknowledgments

The protocol presented here is based on G. Hauptmann’s work (PMID: 11252185), which was implemented and continuously improved by D. Soroldoni. We acknowledge A. Faro and G. Gestri of the Wilson laboratory at UCL, UK who shared the genomic DNA extraction protocol with us. We also thank O. Venzin of the Oates lab for constructive feedback on the manuscript.
  Funding: Institutional support from the Swiss Federal Institute of Technology in Lausanne (EPFL) (to ACO and RN); The Francis Crick Institute (receiving its core funding from Cancer Research UK, the Medical Research Council, and Wellcome) (to ACO and RN); Wellcome (WT098025MA to ACO); the Medical Research Council (MC_UP_1202/3 to ACO and RN).

Competing interests

The authors declare no competing interests.

References

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简介

原位杂交用于可视化组织和胚胎中基因转录物的空间分布,提供有关疾病和发育的重要信息。目前的方法涉及使用掺入非放射性标记的互补核糖核酸探针,其可通过免疫组织化学检测并与生色或荧光可视化相结合。尽管最近的荧光方法已经允许诸如单分子计数的新功能,但由于其相对简单,低成本和高通量以及使用透射光显微镜成像的容易性,定性显色检测对于许多应用仍然是重要的。剩下的挑战是在杂交后将高对比度信号与可靠的基因分型相结合。通常将硫酸葡聚糖添加到杂交缓冲液中以缩短开发时间并改善对比度,但该试剂抑制基于PCR的基因分型。本文描述了使用地高辛(DIG)标记的核糖核酸探针在固定的全寄生斑马鱼胚胎中进行原位杂交的改良方案,该核糖核酸探针用碱性磷酸酶偶联的抗DIG抗体和氮蓝四唑(NBT)/ 5-溴-4-氯-3-吲哚基 - 磷酸(BCIP)显色底物。为了在杂交后产生与下游基因分型相容的胚胎而不牺牲信号的对比度,该方案省略了硫酸葡聚糖并利用较低的杂交温度。
【背景】原位杂交是一种能够检测单个细胞,组织或整个胚胎中RNA的技术(Schulte-Merker 等。,1992; Hauptmann和Gerster,1994 ; Schulte-Merker 等人,1994)。该技术已广泛应用于发育生物学中,以表征固定胚胎中靶mRNA的空间和时间分布(Thisse et al。,2001; Thisse and Thisse,2004和2005)。通过比较野生型,突变体和实验操作的胚胎中的mRNA分布,原位杂交继续在发育中的基因功能研究中发挥重要作用。

杂交基于特定探针寡核苷酸与靶mRNA的互补碱基对结合的原理。设计RNA寡核苷酸(核糖核酸探针)以与靶mRNA分子配对,并与含有半抗原的核苷酸合成,例如洋地黄毒苷(DIG)。在原位杂交中,将核糖核酸探针与先前固定的和透化的大块组织或完整胚胎在升高的温度下孵育,以促进具有高序列互补性的杂交体的形成。然后用半抗原特异性抗体检测稳定的靶 - 探针杂交体。在该技术的显色变异中,抗体与碱性磷酸酶缀合。该酶活性导致在加入生色底物后局部产生有色沉淀物。 NBT和BCIP是常用的底物,产生紫蓝色沉淀。因此,靶mRNA的定位可以通过透射光显微镜中的有色信号在胚胎或组织中用细胞分辨率可视化。

通过在不同探针上包含不同的半抗原(Hauptmann和Gerster,1994)或与免疫组织化学相结合来比较mRNA和蛋白质的定位,已经修改该技术以比较两种mRNA的定位。最近的进展允许荧光检测多种基因(Choi et al。,2010; Gross-Thebing et al。,2014),mRNA的亚细胞定位(Jambor) et al。,2015)和样本中转录本数量的确定(Wang et al。,2012; Little et al。 ,2013; Stapel et al。,2016; Trivedi et al。,2018)。然而,定性显色检测在该领域仍然是重要的,因为其相对简单,低成本,高通量和易于成像。原位杂交实验的有色信号的信噪比是核糖探针特异性的函数,即,核糖探针区分不同靶标的能力,以及杂交的严格性。高严格性需要寡核苷酸中所有碱基的碱基配对,而较低的严格性允许一些碱基未配对。核糖探针的设计设定了杂交的特异性。反应环境影响寡核苷酸的严格退火,并且所得杂交体的热稳定性受温度,pH,一价阳离子浓度和环境中有机溶剂的存在的影响。

核糖核酸探针必须设计成与靶mRNA最大程度地互补,以获得高特异性。另外,最大互补的RNA-RNA杂交体具有最高的热稳定性,因为碱基对错配降低了热稳定性。核糖探针的长度将影响杂交速率并且还影响所得杂交的热稳定性 - 较长的探针具有较高的杂交率并产生热稳定的杂种(Wetmur,1976)。长度在300-3,200碱基对之间的核糖核酸探针可以与本文所述的方案一起使用。

用于杂交的温度由设计的寡核苷酸的解链温度确定。杂交在比计算的解链温度低15-25℃的温度下进行(Wetmur和Davidson,1968) - 该温度越高,杂交的严格性越高。然而,在典型的RNA杂交温度下延长孵育将降低样品形态。因此,添加有机溶剂以降低RNA双链体的热稳定性,甲酰胺是最常用的试剂(McConaughy 等人,1969)。用于原位杂交的杂交缓冲液还含有来自氯化钠和柠檬酸钠的单价钠阳离子,其与RNA-RNA杂合体静电相互作用以稳定它。随后通过优先降低非特异性杂种的稳定性来进行低盐洗涤以减少背景。

用于定性检测斑马鱼中整个mRNA的典型原位方案建议杂交温度为70℃(Thisse和Thisse,2008)以获得高严格性。如果需要核糖探针来区分两个相似的靶序列,则这种高严格条件是必需的。我们发现,对于具有高特异性的核糖核酸探针,通过在较低温度(55-60°C)下进行杂交,我们可以实现更快速发展的高对比度染色。我们可以通过向杂交缓冲液中加入葡聚糖硫酸盐来增加核糖探针的有效浓度,从而进一步缩短开发时间并增强染色的对比度(Lauter et al。,2011)。另外,对于需要延长发育的探针,在NBT-BCIP染色溶液中加入聚乙烯醇会降低背景(Kiyama和Emson,1991)。对原位杂交方案进行这些修改可以持续加速开发并增强染色的对比度。

原位实验的关键部分是对突变体或其他实验操作胚胎中mRNA分布变化的定性分析。然而,在通过改变的形态确定胚胎的基因型之前,mRNA分布的差异可能是明显的。此外,这种差异可能是微妙的或可变的,并且可能不会落入方便的孟德尔比率,这使得野生型和扰动模式的直接分配变得复杂。由于这些原因,在杂交和照相记录之后,具有与胚胎或胚胎片段的可靠基因分型兼容的方案是有价值的。我们观察到杂交缓冲液中硫酸葡聚糖的存在通过PCR抑制基因分型;因此,如果胚胎需要在原位杂交后进行基因分型,则省略。

这里描述的方案描述了三个相关的程序:(1)适合杂交的DIG标记的核糖核酸探针的产生; (2)使用单个核糖核酸探针进行原位杂交,定性检测斑马鱼胚胎发育和胚胎成像中感兴趣的mRNA靶标; (3)DNA提取,其提供PCR-ready提取物,使得能够在先前成像的个体胚胎的基因组DNA中鉴定indel突变的存在或不存在。我们已经在两个不同国家的实验室中成功测试了该协议。结合起来,这些程序形成了足够详细的协议,使相对缺乏经验的实验者能够通过一系列实验扰动实现高质量和可靠的mRNA分布模式分析。

关键字

材料和试剂

  1. Riboprobe合成
    1. 1.5毫升无色微量离心管(Eppendorf,SafeLock,目录号:0030120086)
    2. PCR管(Sarstedt,Multiply ®-μstripPro8-strip,目录号:72.991.002)
    3. 限制性消化酶(Thermo Scientific,FastDigest)(-20°C保存)
    4. 2x Phusion Hot Start II高保真PCR Master Mix(Thermo Scientific,目录号:F-565)(-20°C保存)
    5. rNTP-DIG(罗氏,目录号:11277073910)(-20°C以下的储存)
    6. RNase Inhibitor(RNaseOUT,Invitrogen,目录号:100000840)(-20°C以下保管)
    7. RNA聚合酶(1,000 U-20 U /μl)(-20°C保存)
      1. Sp6(罗氏,目录号:10810274001)
      2. T3(罗氏,目录号:11031163001)
      3. T7(罗氏,目录号:10881767001)
    8. 清理工具包(在室温下存放)
      1. Qiaquick PCR纯化试剂盒(QIAGEN,目录号:28104)
      2. Promega SV凝胶向导PCR纯化试剂盒(Promega,目录号:A9281)
      3. RNeasy Min Elute(QIAGEN,目录号:74204)
    9. 琼脂糖(Sigma-Aldrich,目录号:A9539)(室温下储存)
    10. 1x Tris-Acetate,EDTA(TAE)(在室温下储存,参见食谱)
      1. Tris Base(Sigma-Aldrich,BioXtra,目录号:T6791)(室温下储存)
      2. 冰醋酸(Suprapur ®,EMD Millipore,目录号:1.00066)(室温下储存)
      3. EDTA,0.5 M(UltraPure,Invitrogen,目录号:15575-038)(在室温下保存)
    11. 杂交缓冲液(Hyb)(-20°C储存,参见食谱)
      1. Formamide,deionized(Roche,目录号:1814320)(在4°C下储存)
      2. 50 mg / ml肝素原液(4°C储存,参见食谱)
        肝素(Sigma-Aldrich,目录号:H3400)(室温下储存)
      3. Torula-RNA(Sigma-Aldrich,目录号:R6875)(-20°C保存)
      4. 20x SSC(在室温下储存,参见食谱)
        氯化钠(Sigma-Aldrich,BioXtra,目录号:S7653)
        柠檬酸三钠二水合物(Sigma-Aldrich,目录号:C8532)(在室温下储存)
      5. (可选)葡聚糖硫酸钠盐(Sigma-Aldrich,目录号:D6001)(储存于4°C)
      6. 10%Tween-20(见食谱)
        Tween-20(Sigma-Aldrich,目录号:P9416)(室温下保存)

  2. 胚胎文化
    1. 94毫米聚苯乙烯培养皿(Grenier Bio-One,目录号:633161)
    2. 100x PTU库存(在室温下储存,参见食谱)
      N - 苯基硫脲(PTU,Sigma-Aldrich,目录号:P7629)(在室温下储存)

  3. 原位杂交
    1. 24孔板(Grenier Bio-One,Cell Star,目录号:662160)
    2. 2毫升无色微量离心管(Eppendorf,SafeLock,目录号:0030120094)
    3. 3毫升塑料非无菌,macrograduated巴斯德吸管(HuberLab,目录号:15.4051.11)
    4. 94毫米聚苯乙烯培养皿(Grenier Bio-One,目录号:633161)
    5. 薄壁塑料容器(微波炉塑料饭盒 - 任何品牌)
    6. 甲醇(Fisher,目录号:M / 4000/15)(室温下储存)
    7. 磷酸盐缓冲盐水,0.1%吐温(PBST)(室温下储存,参见食谱)
      1. 氯化钠(Sigma-Aldrich,BioXtra,目录号:S7653)(在室温下储存)
      2. 氯化钾(Sigma-Aldrich,BioXtra,目录号:P9333)(在室温下储存)
      3. 磷酸氢二钠(Sigma-Aldrich,BioXtra,目录号:S7907)(在室温下储存)
      4. 磷酸二氢钾(Sigma-Aldrich,目录号:P9791)(室温下储存)
      5. 10%Tween-20(见食谱)
    8. PBST中4%多聚甲醛(PFA)(储存在4°C)(见食谱)
      1. PFA粉末(Sigma-Aldrich,目录号:P6148)或16%PFA(Alfa Aesar,目录号:43368.9M)(储存在4°C)
      2. PBST(见食谱)
    9. 蛋白酶K库存(-20°C储存,参见食谱)
      Proteinase K(Merck,目录号:1.24568)(在4°C下储存)
    10. 杂交缓冲液(Hyb)(-20°C储存,见食谱)
    11. 杂交后洗涤(见食谱)
      1. 洗1
      2. 洗2
      3. 洗3

  4. 检测
    1. AP-缀合的抗DIG抗体(抗地高辛-AP,来自绵羊的Fab片段)(Roche,目录号:11093274910)(在4℃下储存)
    2. 马来酸缓冲液,0.1%吐温(MABT)(室温下储存,参见食谱)
      1. 马来酸(Sigma-Aldrich,目录号:M0375)(在室温下储存)
      2. 氯化钠(Sigma-Aldrich,BioXtra,目录号:S7653)(在室温下储存)
      3. 氢氧化钠(Fisher Chemical,目录号:S / 4920/60)(室温下储存)
      4. 10%Tween-20(见食谱)
    3. 马来酸缓冲液中含有10%的封闭试剂 
      1. 阻断试剂(罗氏,目录号:11096176001)(室温下保存)
      2. 马来酸缓冲液(见食谱)
    4. MABT中2%阻断剂(2%罗氏块)(见食谱)
    5. 染色缓冲液(见食谱)
      1. Tris-HCl(Sigma-Aldrich,BioXtra,目录号:T6666)(在室温下储存)
      2. 氯化镁六水合物(Sigma-Aldrich,BioXtra,目录号:M2670)(在室温下储存)
      3. 氯化钠(Sigma-Aldrich,BioXtra,目录号:S7653)(在室温下储存)
    6. 染色液(见食谱)
      1. NBT(罗氏目录号:11383213001)(等分试样并在-20°C储存)
      2. BCIP(罗氏,目录号:11282221001)(等分试样并在-20°C储存)
      3. 染色缓冲液(见食谱)
      4. (可选)聚乙烯醇(PVA,Sigma-Aldrich,目录号:P1763)(室温下保存)

  5. 安装
    1. 60毫米培养皿
    2. 人睫毛胶粘在牙签/注射器针头或精细移液器针头上(GELoader Tips0.5-20μl,62 mm,Eppendorf,目录号:0030001222)
    3. (可选)用于整个摄影记录的胚胎模具[模具阴性在我们的实验室制作(Herrgen et al。,2009)]
    平面安装材料
    1. (可选)用钨丝制成的解剖钩(Picker et al。,2009)(Hamilton,目录号:18306)
    2. 10μl移液器吸头 
    3. 玻璃载玻片(Thermo Scientific,Superfrost,目录号:12372098)
    4. 玻璃盖玻片(VWR,MenzelGläser,#1.5矩形22 x 40 mm,目录号:631-1370)
    5. 硅油或凡士林(如凡士林)
    6. 透明指甲油(任何品牌)
    7. 蒸馏水中含87%甘油(室温下储存,参见食谱)
      甘油(Fisher,目录号:G / 0650/08)(室温下储存)

  6. 基因组DNA提取(在室温下保存)
    1. 50x基础解决方案(见食谱)
      1. 氢氧化钾(Fisher Chemical,目录号:P / 5640/60)
      2. EDTA,0.5 M(UltraPure,Invitrogen,目录号:15575-038)
    2. 1x基础解决方案(参见食谱)
    3. 50倍中和解决方案(见食谱)
      Tris-HCl(Sigma-Aldrich,目录号:T6666)
    4. 1x中和解决方案(参见食谱)

设备

  1. 储备溶液制备
    1. 磁力搅拌器(Heidolph Instruments,型号:MR Hei-Standard,目录号:505-20000-00)
    2. pH计(Mettler Toledo,型号:Seven compact和InLab Expert Pro-ISM)

  2. Riboprobe合成
    1. PCR机(Eppendorf,型号:Mastercycler pro S) 
    2. Thermoblock(Eppendorf,型号:ThermoMixer C和SmartBlock 2.0 ml)
    3. 凝胶电泳(Bio-Rad,型号:PowerPac Basic Power Supply;宽Mini-Sub Cell GT Cell)
    4. NanoDrop分光光度计(Thermo Fisher,型号:NanoDrop TM 2000,目录号:ND 2000)

  3. 原位杂交
    1. 镊子(Dumont,型号:5-Inox-E)
    2. 立体显微镜(奥林巴斯,型号:SZ61)
    3. Shaker(Heidolph,型号:Duomax1030,目录号:543-32205-00)
    4. (可选)真空设置(Integra,Vacusafe吸气系统)
    5. 水浴(GFL,型号:1003)
    6. (可选)井板支架(Diversified Biotech,目录号:WPST-1000)

  4. 文档
    1. RGB相机(奥林巴斯,型号:DP22)
    2. 环形灯(光子光学,型号:LED 40环形灯)

  5. 基因组DNA提取
    1. 微刀(精细科学工具,目录号:10315-12)

软件

  1. 斐济(Schindelin et al。,2012, https://fiji.sc/
  2. Snapgene(GSL Biotech LLC)

程序

  1. Riboprobe合成
    注意:用于核糖核酸探针的DNA模板可以克隆到RNA聚合酶启动子下游的质粒骨架中,或者从cDNA或基因组DNA扩增的PCR产物中克隆。见注1。
    1. 模板准备
      从质粒准备模板
      1. 通过用合适的限制酶在37℃消化使质粒线性化。选择一种酶,使模板在cDNA插入物的下游直接线性化。
        将反应设置为:
        class =“ke-zeroborder”bordercolor =“#000000”style =“width:200px;” border =“0”cellspacing =“0”cellpadding =“2”>质粒模板
        5μg
        限制酶
        5μl
        10x限制缓冲区
        10μl
        用蒸馏水/无核酸酶水补足至100μl。
      2. 用商业清理试剂盒纯化线性化质粒。我们推荐使用Qiaquick PCR纯化试剂盒。按照制造商的说明纯化质粒,并在30μl洗脱缓冲液中洗脱质粒。
      3. 在4μl蒸馏水中稀释1μl洗脱液。使用1μl评估分光光度计上的浓度和4μl,以检查琼脂糖凝胶电泳的线性化。在凝胶上应该可以看到对应于线性化质粒的单一条带。如果可视化对应于完全线性化质粒的单一条带,则继续进行riboprobe合成。 

      通过PCR准备模板
      1. 设计引物以从cDNA或基因组DNA扩增所需的靶标。为了制备与靶mRNA互补的核糖核酸探针,将RNA聚合酶(T7 / T3 / Sp6)启动子序列添加到反向引物中:5'-RNA聚合酶启动子序列+模板特异性序列-3'。


      2. 在最佳循环条件下进行PCR。我们建议使用像Phusion这样的高保真DNA聚合酶。
      3. 清理PCR产物。我们推荐使用Promega SV凝胶向导PCR纯化试剂盒。在30μl试剂盒洗脱缓冲液中洗脱。在4μl蒸馏水中稀释1μl洗脱液。使用1μl此稀释液评估NanoDrop和4μl浓度,通过凝胶电泳评估产品质量。如果获得适当长度的单个条带,则继续进行riboprobe合成。没有必要对PCR产物进行测序以确认模板的序列准确性(参见注释6)。
    2. 体外转录
      1. 解冻反应组分(酶除外)并置于冰上。
      2. 将每个20μl转录反应设置为:
        class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“9”>DNA模板
        1-1.5μg线性化质粒
        或100-500 ng的PCR产物
        10x转录缓冲液
        2μl
        rNTP-DIG
        2μl
        RNase抑制剂
        0.5μl
        RNA聚合酶
        2μl
        如果需要,使用不含RNase的水将体积调节至20μl。
      3. 在37°C转录2-4小时。当转录时间在2小时和4小时之间时,反应的产率是相等的。
      4. 用QIAGEN RNeasy Min Elute等商业试剂盒从反应中纯化核糖核酸探针。按照制造商的说明,用30μl不含RNase的水洗脱核糖核酸探针。在4μl水中稀释1μl探针。使用1μl评估分光光度计上的浓度和4μl,以通过琼脂糖凝胶电泳评估转录质量。清理后合成探针的典型A 280 / A 260 比率在1.85和2.00之间。为了在1x TAE中的1%琼脂糖凝胶上解析RNA核糖探针,在140V下运行10-15分钟。核糖探针应该作为一个独特的条带解析。偶尔也会获得更长的条带 - 如果使用高质量的PCR产物或完全线性化的质粒模板,这通常是由RNA二级结构引起的电泳伪影。
      5. 将合成的核糖探针的剩余部分储存在-20℃。在5ml Hyb中稀释核糖探针以进行原位杂交。见注2。
      6. 通过进行原位杂交测试探针并评估获得的杂交模式。 

  2. 原位杂交
    注意: 
    1. 该方案针对受精后48小时(hpf)的胚胎进行了优化,并在24孔板中进行。使用800μl进行洗涤,使用500μl进行孵育。在更换培养基时,不应让被染色的胚胎变干,并且应始终保持液体覆盖。为了确保它们不会粘在孔的底部或侧面,轻轻移液并在每次溶液更换后检查胚胎是否被液体覆盖。
    2. 除非另有说明,所有孵育步骤均在室温下在振荡器上进行。

    1. 胚胎的固定和脱水(见注3)。
      修复24 hpf以下的胚胎
      1. 使用3ml塑料巴斯德吸管将胚胎转移到2ml微量离心管(用于长期储存)或24孔板的孔中(立即用于原位)。一个管中可存储多达100个胚胎,24孔板中每孔可处理多达30个胚胎。
      2. 去除尽可能多的胚胎培养基。如果胚胎在绒毛膜中,则可以去除所有液体。在PBST中加入4%PFA(固定)每管-1ml,每孔800μl。在室温下在振荡器上孵育5分钟。该步骤用于去除任何多余的胚胎培养基。
      3. 用新鲜固定剂将固定物更换为每管2ml,每孔800μl-1ml。在室温下孵育1至4小时或在4℃下振荡过夜。
      4. 取出固定液,每管加入2毫升PBST,每孔800μl-1 ml。
      5. 使用3ml塑料巴斯德吸管将胚胎转移到35mm培养皿中的PBST中,并使用镊子在立体显微镜下对胚胎进行去离子化(视频1)。将胚胎移回孔板或新鲜管/小瓶中。应通过在PBST中上下移液来冲洗移液管,并在批次胚胎之间更换PBST以防止交叉污染。 

      固定胚胎超过24 hpf
      1. 从24 hpf开始,将PTU添加到胚胎培养基中至终浓度为0.003%,以抑制胚胎中的色素发育。
      2. 在立体显微镜(视频2)下在35mm培养皿中的胚胎培养基中脱落胚胎,并用3ml塑料巴斯德吸管将它们转移至孔板或微量离心管小瓶-30用于孔,100μL用于管。应在批次胚胎之间交换胚胎培养基以防止交叉污染。
      3. 去除尽可能多的胚胎培养基。在PBST中加入4%PFA(固定)每管-1ml,每孔800μl。在室温下在振荡器上孵育5分钟。该步骤用于去除任何多余的胚胎培养基。
      4. 用每个管或小瓶2ml的新鲜固定物进行交换,在孔板中每孔800μl至1ml。在室温下振荡孵育1至4小时或在4℃下过夜。
      5. 通过移除固定物并将PBST加入每管2ml,每孔800μl-1ml,简单地冲洗胚胎。
      6. 交换PBST并在室温下在摇床上在PBST中洗涤胚胎3次,每次持续5分钟。

      胚胎的脱水和透化
      1. 在分级甲醇系列中使胚胎脱水:在PBST中加入800μl30%甲醇,然后用PBST中的800μl50%甲醇替换,然后用PBST中的800μl70%甲醇替换。在每次更换之间孵育振荡器5分钟。
      2. 在振荡器上将胚胎在800μl无水甲醇中孵育5分钟。
      3. 更换甲醇并在-20℃下孵育胚胎过夜以使胚胎透化。每孔使用1.1毫升甲醇或每微升管2毫升。
        如果需要,可以在此处停止该方案,因为胚胎可以无限期地在-20°C的甲醇中保存(见注3)。


        视频1


        视频2


      视频1.固定的发育阶段胚胎的脱离
      视频2.受精(hpf)胚胎后36小时活体脱落

    2. 酶胚透化
      1. 在分级甲醇系列中再水化胚胎:在PBST中加入800μl70%甲醇,然后用PBST中的800μl50%甲醇替换,然后用PBST中的800μl30%甲醇替换。每个步骤在振荡器上孵育5分钟。 
      2. 用PBST进行两次5分钟的洗涤。
      3. 消化蛋白酶K(PKA)中的胚胎。对于小于24 hpf的胚胎,1:200稀释PKA原液,对于PBST中早于24 hpf的胚胎,1:100稀释PKA原液。在室温下不振荡孵育500μl。消化时间依赖于胚胎期;下面列出的时间可以作为指导。这里指出的阶段是根据Kimmel 等人(1995)。



      4. 通过与PBST快速交换然后在室温下在摇床上用500μl固定物重新固定20分钟来停止反应。
      5. 删除修复;用PBST进行一次快速洗涤,然后进行两次5分钟的PBST洗涤。
    3. 杂交
      注意:用于杂交的温度取决于探针,但良好的起始温度为60°C(见注4)。在设定为杂交温度的水浴中进行杂交步骤。使用内衬有湿纸巾的薄壁塑料容器在水浴中孵育孔板。容器和杂交溶液需要在使用前预热至水浴中的杂交温度。
      1. 在杂交温度下在500μlHyb中预杂交至少30分钟。胚胎可以在杂交温度下预杂交3小时。 
      2. 取出Hyb并用500μl用Hyb稀释的标记核糖核酸探针替换。在杂交温度下杂交过夜。见注5。
    4. 杂交后洗涤
      注意:这些洗涤也在杂交温度的水浴中进行。准备好清洗液并在使用前将它们在水浴中加热。此步骤中显示的计时非常重要 - 请勿在比建议时间更短或更长的时间内进行清洗。
      1. 取下并回收探头。储存在-20°C。见注2。
      2. 在洗涤1中每次洗涤胚胎2次,每次30分钟。
      3. 在洗涤2中洗涤胚胎15分钟。
      4. 在Wash 3中每次洗涤胚胎2次,每次30分钟。
    5. 检测
      1. 用MABT快速冲洗,从胚胎中取出剩余的Wash 3。
      2. 加入500μl2%封闭试剂,在室温下避光孵育至少30分钟。
      3. 在2%封闭试剂中稀释AP缀合的抗DIG抗体1:2,000。取出封闭试剂,在封闭试剂中加入500μl稀释的抗体至胚胎中。在室温下避光孵育2小时,不摇晃。
      4. 取出抗体溶液,用MABT快速洗2次。
      5. 在室温下以1至2小时的间隔洗涤10至20分钟;至少应该进行6次MABT交换。或者,胚胎可以在4℃下在MABT中洗涤过夜,然后在第二天洗涤3次,每次5分钟。
      6. 将胚胎在新制备的染色缓冲液中平衡两次,每次5分钟。
      7. 向胚胎中加入500μl染色溶液。在室温下在黑暗中染色而不摇动。见注5。
      8. 在立体显微镜下通过眼睛控制染色反应。将孔板放在白色背景上,从上方照亮板。在加入染色溶液后的前5分钟监测胚胎的发育,然后以10-15分钟的间隔监测胚胎的发育,直到染色剂饱和或达到所需的对比度。
      9. 通过在PBST中进行三次5分钟的洗涤来停止染色反应。
      10. 在室温下将胚胎在甲醇中孵育直至蛋黄被清除并呈现白色,或在4°C或-20°C过夜。
        注意:有关我们使用此协议制作的污渍,请参见图2,3B和4B。有关故障排除的说明,请参见注释。

    6. 安装和摄影
      1. 通过在PBST中进行两次5分钟的洗涤,从胚胎中除去甲醇。
      2. 在室温下在PBST中重新固定4%PFA至少15分钟。
      3. 在PBST中洗涤胚胎两次,每次5分钟。
      4. 向胚胎中加入87%甘油,并在4℃下平衡。当胚胎下沉到井底时,它们完全平衡。这需要约12小时。随后可将胚胎在4°C或-20°C下储存在87%甘油中。
      5. 拍摄胚胎: 
        1. 拍摄整个装备:
          使用耦合到立体显微镜的RGB相机在35mm培养皿或成像模具(参见注释7)中将胚胎拍摄在87%甘油中。使用环形灯从上方照亮胚胎。在碟子下面放一张白纸,为图像提供白色背景。可以使用睫毛,钨丝或精细移液器尖端来操纵胚胎。 
        2. 拍摄平板准备:
          另请参阅用于去除和平面安装的JoVE视频协议(Cheng et al。,2014)。
          1)
          将胚胎平放在带有一滴87%甘油的载玻片上。用滴眼液中的睫毛或尖锐的钨丝(参见注释8)对胚胎进行脱蛋。将胚胎移到液滴的边缘,使最小量的甘油覆盖它。用睫毛去除剩余的卵黄细胞。定期从甘油滴中的胚胎中冲洗松散的卵黄细胞。去除后,将胚胎移至新鲜的甘油滴中。
          2)
          将背侧的胚胎轴放在干净的玻璃载玻片上,用金属丝或睫毛夹住。将约15-20μl总体积的87%甘油置于胚胎旁边的小滴中。最多10个去卵黄胚胎可以平放在一个载玻片上。
          3)
          用10μl移液管尖端制作4个小油脂柱,将玻璃盖玻片的角部固定到位。小心地将盖玻片放在载玻片上(图1)。
          4)
          用透明指甲油密封盖玻片的4个边缘。
          5)
          使用RGB相机在立体镜上用透射光拍摄胚胎。

          图1.准备平底座。 A.将甘油滴放在去卵黄的胚胎旁边(带圆圈),然后轻轻地放下载玻片上的盖玻片,盖玻片将被角落处的油脂柱固定到位。 B.带有扁平安装胚胎(带圆圈)的完成的载玻片和用透明指甲油密封的盖玻片边缘。

  3. 基因分型
    注意:原位杂交后,可从完整胚胎或组织碎片中提取基因组DNA。在去蜕皮和扁平安装胚胎之前取出组织碎片。每管在PCR管-1胚胎中执行以下步骤。
    1. 选择要进行基因分型的胚胎,并在小培养皿中一起冲洗,或者在含有PBST的PCR管中单独冲洗以除去甘油。几次快速交换,然后在室温下在振荡器上进行三次5分钟的洗涤通常就足够了。
    2. 用镊子将整个胚胎转移到PCR管中。如果从组织碎片中提取DNA,则将胚胎转移到载玻片上,进入15-20μl的PBST滴。切割三分之一至二分之一未用微刀染色的胚胎。将片转移到PCR管中。确保没有多余的液体转移。在载玻片上的组织中加入87%甘油,继续进行脱色和平面安装。 
    3. 向管中加入15μl1x碱溶液,在95°C下孵育30分钟。
    4. 升至室温并加入15μl1x中和溶液。
      注意:此解决方案已准备好PCR(参见注释9)。

数据分析

  1. 与生色检测偶联的整体mRNA 原位杂交是胚胎中基因表达的定性测定。在整个装载形式中,可以评估感兴趣的发育阶段中特定组织中感兴趣的mRNA的分布。对固定在不同发育阶段的胚胎中的靶标进行mRNA 原位杂交可以帮助研究者表征在发育过程中感兴趣基因的mRNA表达的时间变化。
    原位杂交也可用于评估基因功能的丧失或敲低对目的基因表达的影响。例如,原位杂交已经在我们的实验室中用于测定在发育过程中斑马鱼胚胎中它们的转录振荡引起的环状基因的波形,并证明功能突变体环基因等位基因的丧失导致这些波浪模式的丧失(图1,Lleras Forero 等人,2018)。利用一组胚胎显微镜图像,原位杂交也可以用作半定量终点分析来测量实验扰动的时空效应,例如调查上调或Wnt信号传导的下调(图3,5和6,Bajard 等人,,2014)。 mRNA 原位杂交也被用于进行内源性环基因表达的空间分布与转基因报告基因之间的比较(图S1,Soroldoni et al。,2014) 。但是,必须注意在定性上解释依赖于生色信号强度的结果,或者在用斐济(https://fiji.sc/)中的绘图轮廓等工具量化信号强度之后,因为染色的强度最初是发育时间依赖性的,但酶促反应可以随后饱和。
  2. 从提取的基因组DNA设计基因分型策略将完全取决于实验中特定等位基因的性质。对于一些等位基因,碱基转换将导致限制性消化的位点的获得或丧失,允许使用对目的基因特异的引物从基因组DNA获得的PCR扩增子的基于限制性消化的分析。当这种策略不可能时,可以对PCR扩增子进行测序。产生的基因分型序列数据的分析可以用开源或市售软件(Snapgene,GSL Biotech LLC)完成。例如,我们在 her7 hu2124 背景最初通过测序(图S1,Lleras Forero 等人,2018)。该突变导致限制性位点的丧失,随后通过 her1 突变体等位基因对限制性消化的抗性对胚胎进行基因分型。

笔记

  1. Riboprobe设计
    通常,使用长探针可提供良好的染色效果。长探针产生具有高热稳定性的杂交体并提供更大的信号放大。因此,一种良好的通用方法是设计与mRNA的整个编码序列互补的核糖探针。但是,这种方法可能并不适用于所有情况。例如,为了区分旁系同源物的mRNA,设计与感兴趣的旁系同源物具有高互补性的较短的核糖核酸探针是更好的方法。在我们的手中,使用该方案可以可靠地产生大约300-3,200bp长度的探针(图2)。 


    图2. 原位杂交方案的代表性结果,使用长度约为300和3,200碱基对的核糖探针。 A. paraxial protocadherin 的染色 papc / pcdh8 )使用3,137碱基对长度的核糖探针,与 papc mRNA的编码区反义(Yamamoto et al。,1998) 。 B.使用325碱基对核糖探针染色 ripply1 ,与 ripply1 mRNA的编码区反义(Lleras 等,,2018)。在10℃的野生型胚胎上在60℃下进行杂交。这里示出了平面安装的制剂,其朝向面板的底部定向。比例尺为50μm,适用于两个面板。 

  2. 探针浓度
    探针的最终工作浓度应通过实验优化。通常,浓度为0.3-1 ng /μl会产生良好的原位染色;但是,每次合成新探针时都应滴定工作浓度。探头可以循环使用多次。记下初始体积,因为探针会随着时间的推移而稀释,并且不会产生高对比度污渍。
  3. 胚胎固定和储存
    胚胎可以提前固定和脱水。修复的质量将影响污渍的信噪比。新制备的修复程序可以获得最佳效果,但可以使用长达一周的修复程序。胚胎不应在室温下固定超过4小时或在4°C下固定1周。固定和脱水的胚胎可以无限期地在-20°C的甲醇中储存。
  4. 杂交温度
    杂交温度取决于探针,必须通过实验确定每种探针。温度的选择也决定了方案的严格性 - 严格性可以通过提高杂交温度来提高,并通过降低它来降低。当使用需要区分具有高序列相似性的靶标的探针时,需要提高方案的严格性以有利于形成所需的RNA杂交体,因为1%碱基对错配通过粗略降低RNA-RNA双链体的热稳定性。 1°C(Wetmur et al。,1976)。
    在我们的手中,设计用于检测靶mRNA编码序列的探针的良好起始温度为60℃(图2和3)。对于短探针和内含子探针,55°C已用作起始温度。然后通过以2°C的步长改变温度来优化杂交温度。
     

    图3. 60°C杂交足够严格,可通过特异性探针获得高对比度的最终染色。 原位杂交检测 xirp2a (Deniziak et al。,2007)在70℃和60℃下在36hpf野生型胚胎中进行。 A.当探针在70℃杂交时,显影110分钟后染色。 B.当在60℃杂交时,显影50分钟后染色。在杂交缓冲液中使用的核糖核酸探针浓度为0.5ng /μl。比例尺为100μm,适用于两个面板。

  5. 改善污渍的对比度
    与不使用葡聚糖硫酸盐的污渍相比,向杂交缓冲液中加入硫酸葡聚糖将产生具有相对短的显影时间的高对比度污渍(图4)。如果需要较长的开发时间来检测目标mRNA,向染色溶液中加入10%PVA会降低背景染色的发生速率。


    图4.向杂交缓冲液中加入硫酸葡聚糖可增强最终染色的对比度。 原位杂交检测 xirp2a 在60°进行C在36 hpf野生型胚胎中。 A.当探针在杂交缓冲液B中杂交后显影25分钟后染色。在探针与含5%硫酸葡聚糖(DS)的杂交缓冲液中杂交后,在显色25分钟后染色。使用的核糖核酸浓度为1 ng /μl。比例尺为100μm,适用于两个面板。

  6. 故障排除
    没有来自反义核糖探针的信号
    体外转录尚未成功:质粒的完整性可能受到损害。重新制备质粒或通过PCR生成模板。

    次优信噪比
    1. 质量差的探头:通过模板和合成的核糖核酸探针的琼脂糖凝胶电泳来控制探针质量。他们应该解决不同的乐队。可以对模板进行排序以评估序列准确性。
    2. 探头浓度过高:稀释探头会降低背景。
    3. 背景由于非特异性杂交:通过提高杂交温度来提高方案的严格性。
    4. 背景由于延长的发展:降低杂交温度以降低方案的严格性,向染色溶液中加入10%PVA。

    信号强度差
    1. 探针太稀:合成新探针,增加探针浓度。
    2. 探针设计:增加探针长度,以获得更好的信号放大。
    3. 杂交过于严格:降低杂交温度。
    4. 向杂交缓冲液中加入5%硫酸葡聚糖。 
  7. 整体装备摄影模具
    用于拍摄整个样品的成像皿可以通过使用聚二甲基硅氧烷(PDMS)阴性在1-1.5%琼脂糖的蒸馏水中浇铸胚胎的模具来制备。这种负片是由有机玻璃制成的正片加工而成的,所述塑料机械加工有所需直径和深度的圆锥形凹陷(图4B,成像模具的正面和负面由我们的实验室制作,并在Herrgen 等人中描述。 ,2009)。向培养皿中加入87%甘油。将染色的胚胎转移到模具中,并使用钨丝,睫毛或精细移液管尖端将其定向,使其蛋黄适合立体显微镜下的锥形凹陷(图5)。


    图5.准备记录整个原位杂交制剂 使用PDMS阴性,其具有0.8 mm直径和0.4 mm高度的凸起锥体阵列用于投射琼脂糖模具有锥形凹陷。胚胎的蛋黄适合凹陷,胚胎可以横向定向,使得它们平放在琼脂糖的表面上。 A.铸造模具以使胚胎在1%-1.5%琼脂糖中定向。 B.胚胎在87%甘油中定向在模具的锥形腔中。 C-C”。聚二甲基硅氧烷(PDMS)阴性用于铸造模具。 D.胚胎的琼脂糖模具。 E.面向摄影的胚胎。 

  8. 去除胚胎
    1. 我们建议参考视频协议进行去抖动和平面安装(Cheng et al。,2014)。
    2. 可以使用尖锐的钨丝或“钩”代替睫毛用于去除胚胎。为了锐化和塑造电线,在本生灯火焰中保持最后0.5至1厘米长的电线,直到它发出橙色30-60秒,然后对钨丝末端进行抛光。用一对镊子将火焰线形成87%甘油的钩子。 Picker 等人,2009中提供了制作钨钩和支架的详细说明。
  9. 使用胚胎提取物进行PCR
    应该试验在蒸馏水中不稀释至1:5的稀释系列,以找到在给定胚胎阶段PCR所必需的基因组DNA提取物的最佳体积。从8到10个体节阶段的整个胚胎和胚胎片段的提取物通常不需要任何进一步稀释,并且每10μlPCR反应可以使用1-2μl。 24-36 hpf胚胎组织的提取物通常需要1:4稀释。

食谱

注意:本协议不要求DEPC处理过的水或溶液或特定的无RNase条件。蒸馏或超纯水,高压灭菌玻璃器皿和一次性聚丙烯消耗品,如微量离心管,离心管,塑料巴斯德吸管微量吸管尖端就足够了。

  1. 1x TAE
    40 mM Tris-acetate
    20 mM醋酸盐
    1 mM EDTA
    pH 8.3
    用超纯水稀释10倍TAE 1:10
    在室温下储存
    制备1 L 10x TAE(0.4 M Tris-acetate,0.2 M Acetate和0.1 M EDTA):
    48.5克Tris碱
    11.4毫升冰醋酸
    20ml 0.5M EDTA,pH 8.0
    用超纯水补足1升
    高压灭菌器
  2. 100x PTU原液(蒸馏水中0.3%[重量])(100ml)
    在加热和搅拌下将0.3g N - 苯基硫脲(PTU)溶于100ml蒸馏水中(在室温下避光保存)
  3. 10%吐温-20(在蒸馏水中稀释吐温-20:1)(100ml)
    10毫升吐温-20
    用蒸馏水补足至100毫升
    搅拌直至达到均匀的溶液
    在室温下储存
  4. PBST
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    蒸馏水中的1.8 mM KH 2 PO 4 ,pH 7.4
    0.1%Tween-20
    制作1 L PBST:
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24克KH 2 PO 4
    调节pH值并高压灭菌后,用蒸馏水补足至1L
    加入10毫升10%Tween-20
    在室温下储存
  5. PBST中4%PFA(PBST中4%重量)(100ml)
    1. 在水浴中预热100 ml PBS至68°C
      加入4克PFA
      在68°C孵育,定期搅拌50-60分钟
      当溶液冷却至室温时,加入1ml 10%Tween-20 在4°C下储存长达一周
      注意:PFA应在50分钟至1小时内溶解
      要么
    2. 在PBST中稀释16%PFA 1:4。在4°C下储存长达一周
  6. 蛋白酶K库存
    1毫克/毫升蒸馏水
    在-20°C下保存等分试样
  7. 20x SSC
    3 M NaCl
    300 mM柠檬酸三钠
    pH 7.0-7.2
    制作1 L 20x SSC:
    175.2克NaCl
    88.2克柠檬酸三钠
    调节pH后,用蒸馏水补足1升
    高压灭菌器
    在室温下储存
  8. 50毫克/毫升肝素原料
    在蒸馏水中溶解在肝素中至终浓度为50mg / ml
    储存在4°C
  9. 杂交缓冲液(Hyb)
    50%Formamide
    5x SSC
    50μg/ ml肝素
    0.5 mg / ml Torula-RNA
    0.1%Tween-20
    可选:5%体积的葡聚糖硫酸盐
    制作50毫升Hyb:
    25毫升甲酰胺
    12.5毫升20倍SSC
    50μl肝素原料
    25毫克Torula-RNA
    2.5克葡聚糖硫酸盐
    涡旋很好,直到达到均匀状态
    加入500μl10%Tween-20
    用蒸馏水补足至50毫升
    储存在-20°C
    注意:用于预杂交的hyb中省略了硫酸葡聚糖。
  10. 杂交后洗涤
    洗1: 50%甲酰胺,2x SSC,0.1%Tween-20蒸馏水
    制作50毫升:
    25毫升甲酰胺
    5毫升20倍SSC
    500μl10%Tween-20
    用蒸馏水补足至50毫升
    洗涤2: 2x SSC,0.1%吐温-20在蒸馏水中
    制作50毫升:
    5毫升20倍SSC
    500μl10%Tween-20
    用蒸馏水补足至50毫升
    洗涤3: 0.2x SSC,0.1%吐温-20在蒸馏水中
    制作50毫升:
    500μl20xSSC
    500μl10%Tween-20
    用蒸馏水补足至50毫升
    注意:将适量的清洗液新鲜。
  11. MABT
    100 mM马来酸
    蒸馏水中含150 mM NaCl
    pH 7.5
    0.1%Tween-20
    制作1 L:
    8.77克马来酸
    11.6克NaCl
    用NaOH颗粒调节pH值 用蒸馏水补足1升
    高压灭菌器
    加入10毫升10%Tween-20
    在室温下储存
  12. 10%封闭试剂(罗氏块)
    按照制造商的说明在MAB中制备10%封闭试剂
    在-20°C下保存,等分试样
  13. MABT中2%阻断剂(2%罗氏块)
    在MABT中以1:5稀释10%封闭试剂(罗氏嵌段)
    新鲜
  14. 染色缓冲液
    100mM Tris-HCl,pH 9.5
    100 mM NaCl
    蒸馏水中的50mM MgCl 2,
    制作50毫升:
    5毫升1M Tris-HCl,pH 9.5
    5毫升1M NaCl
    2.5毫升1M MgCl 2
    500μl10%Tween-20
    注意:新鲜。
  15. 染色液
    每1ml染色缓冲液中含4.5μlNBT和3.5μlBCIP
    Vortex混合
    避光保存
    注意:准备适当的音量。
    可选:添加PVA至终浓度10%,体积重量
    用10%PVA制备10 ml染色液:
    将1g PVA加入10ml染色缓冲液(不含吐温-20)中 通过搅拌和加热进入溶液 冷却至室温
    加入45μlNBT和35μlBCIP
    Vortex混合
    避光保存
    注意:10毫升是可以制作的最小音量。
  16. 蒸馏水中87%甘油(重量)
    制作100克:
    在13克蒸馏水中加入87克甘油
    搅拌直至获得均匀的溶液
    在室温下储存
  17. 50倍基础解决方案
    1.25 M KOH
    蒸馏水中的10 mM EDTA
    制作200毫升:
    14克KOH
    4毫升0.5毫升EDTA
    加入蒸馏水至终体积200毫升
    在室温下储存
  18. 1x基础解决方案
    用蒸馏水稀释50倍碱溶液1:50
    注意:准备适当的音量。
  19. 50倍中和解决方案
    在蒸馏水中加入2 M Tris-HCl
    制作200毫升:
    63克Tris-HCl
    加入蒸馏水至终体积200毫升
    在室温下储存
  20. 1x中和解决方案
    在蒸馏水中稀释50倍中和溶液1:50
    注意:准备适当的音量。

致谢

这里介绍的协议基于G. Hauptmann的工作(PMID:11252185),由D. Soroldoni实施并不断改进。我们感谢英国伦敦大学学院威尔逊实验室的A. Faro和G. Gestri,他们与我们分享了基因组DNA提取方案。我们还要感谢Oates实验室的O. Venzin对手稿的建设性反馈。
 资金来源:瑞士洛桑联邦理工学院(EPFL)(ACO和RN)提供的机构支持;弗朗西斯克里克研究所(从英国癌症研究中心,医学研究委员会和威康获得核心资金)(ACO和RN);惠康(WT098025MA到ACO);医学研究委员会(AC_和RN的MC_UP_1202 / 3)。

利益争夺

作者宣称没有竞争利益。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Narayanan, R. and Oates, A. C. (2019). Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos. Bio-protocol 9(6): e3193. DOI: 10.21769/BioProtoc.3193.
  2. Lleras Forero, L., Narayanan, R., Huitema, L. F., VanBergen, M., Apschner, A., Peterson-Maduro, J., Logister, I., Valentin, G., Morelli, L. G., Oates, A. C. and Schulte-Merker, S. (2018). Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock. Elife 7: e33843.
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