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The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
RiboPuromycylation方法(RPM):一种在亚细胞水平绘制翻译位点图谱的免疫荧光技术   

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The Journal of Cell Biology
Apr 2012

Abstract

While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.

Keywords: Translation site (翻译位点), Puromycin (嘌呤霉素), Ribopuromycylation (Ribopuromycylation), Ribosome (核糖体), Nascent chain (新生链)

Background

For decades, isotopic labeling of amino acids has been considered as the gold standard for studying protein translation. Though this method has proven to be remarkably accurate for evaluating translation rates, it provides no information on the location of translating ribosomes. More recently, amino acids analogs have enabled fluorescent detection nascent chains (Dieterich et al., 2007). Nevertheless, nearly all of the detected signal comes from polypeptides released from ribosome. Our initial idea was to develop a method to label nascent chains while still tethered to translating ribosomes.

Puromycin (PMY) is an aminoglycoside antibiotic that mimics charged tRNATyr and incorporates into the ribosome A site. Consequently, PMY triggers premature translation termination by ribosome catalyzed-covalent incorporation into the nascent chain COOH-terminus (Pestka, 1971) followed by release of PMY-peptide. Polyclonal antibodies (Abs) to PMY were initially generated (Eggers et al., 1997) to detect puromycylated nascent chains released from ribosomes by immunoblotting and immunoprecipitation. Subsequently, fluorescent PMY was used to label nascent chains by microscopy (Starck et al., 2004). Schmidt et al. (2009) found that cells exposed to PMY generate a sufficient amount of PMY-terminated cell surface proteins to enable detection by live cell flow cytometry using a monoclonal Ab (mAb), providing a measure of translation rates. Importantly, none of these methods discriminate between ribosome-attached or released PMY-peptides and are all hindered to some extent as measures of translation by degradation of released proteins.

Having found that chain elongation inhibitors, such as cycloheximide (CHX) or emetine, prevent the release of PMY-nascent chain from ribosomes, we developed the ribopuromycylation method (RPM) that enables immunofluorescent detection of translation sites at the sub-cellular level (Figure 1) (David et al., 2011 and 2012b). This method has been used by our labs and many other labs to study translation in neurons (Biever et al., 2015; Perry et al., 2016; Williams et al., 2016), migrating cells (Willett et al., 2011), immune cells (Seedhom et al., 2016) and stressed or infected cells (Kedersha et al., 2016; Emmott et al., 2017; Roth et al., 2017).


Figure 1. Schematic representation of RPM (from David et al., 2012b). Following freezing of polysome with an elongation inhibitor (step 1), PMY is added (step 2) to living cells and nascent chains become puromycylated through ribosome catalysis (step 3). Anti-PMY monoclonal antibodies detect puromycylated nascent chains via indirect immunofluorescence (step 4). Reproduced from David et al. (2012b) with permission of the publisher and of Dr. Yewdell.

Materials and Reagents

  1. Materials
    1. 6-well and 24-well plates (Corning, Costar®, catalog numbers: 3506 , 3524 )
    2. High quality glass coverslips, 12 mm diameter, #1 thickness (Glaswarenfabrik Karl Hecht, Assistent, catalog number: 1001/12 )
      Note: Either autoclave or sterilize with 70% ethanol before use.
    3. Microscope slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: J1800AMNZ )
    4. Whatman paper (GE Healthcare, catalog number: 3030-917 )
    5. Parafilm (Bemis, catalog number: 701606 )
    6. Petri dishes (Corning, catalog number: 430591 )
    7. Aluminum foil (Sigma-Aldrich, catalog number: Z691569 )
      Manufacturer: Heathrow Scientific, catalog number: HD23534A .
    8. Plastic tips
  2. Cell line(s)
    The described procedure and associated figure (Figure 2) uses HeLa cells (ATCC, catalog number: CCL-2.1 )
    Note: Though this procedure was originally designed for HeLa cells, it can be easily adapted for other adherent cell lines (Graber et al., 2013). However, controls with several inhibitors are needed to validate any adjustment (e.g., Digitonin concentration) of any kind. For non-adherent cells, we describe an alternate protocol (Procedure D).
  3. Reagents
    1. Alcian blue (Sigma-Aldrich, catalog number: A5268 )
    2. 70% ethanol (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R40135 )
    3. Hoechst 33258 (Thermo Fisher Scientific, InvitrogenTM, catalog number: H1398 )
    4. Fluoromount-G (SouthernBiotech, catalog number: 0100-01 )
    5. Potassium phosphate monobasic (KH2PO4)
    6. Sodium chloride (NaCl)
    7. Na2HPO4·7H2O
    8. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41966029 )
    9. Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
    10. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
    11. Fetal bovine serum (FBS) (Eurobio, catalog number: CVFSVF0101 )
    12. Digitonin (Wako Pure Chemical Industries, catalog number: 043-21376 )
    13. Tris-HCl pH 7.5 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15567027 )
    14. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M0250 )
    15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    16. Complete Mini EDTA-free protease inhibitor tablets (Sigma-Aldrich, Roche Diagnostics, catalog number: 11836170001 )
    17. RNase Out (Life Technologies, catalog number: 100000840 ; or Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
    18. DEPC treated water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 750023 )
    19. 16% paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15710 )
    20. Sucrose (Sigma-Aldrich, catalog number: 84097 )
    21. Saponin (Sigma-Aldrich, catalog number: 84510 )
    22. Glycine (Sigma-Aldrich, catalog number: G7126 )
  4. Protein synthesis inhibitors (see Table 1)
    1. Anisomycin (Sigma-Aldrich, catalog number: A9789 )
    2. Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
    3. Emetine dihydrochloride (Sigma-Aldrich, catalog number: E2375 )
    4. Harringtonine (Santa Cruz Biotechnology, catalog number: sc-204771 )
    5. Puromycin (PMY) (Sigma-Aldrich, catalog number: P7255 )
    6. Sodium arsenite (NaAsO2) (Sigma-Aldrich, catalog number: S7400 )
  5. Antibodies
    1. Primary antibodies
      1. Anti-PMY mouse monoclonal antibodies: We tested 3 mouse mAbs from different hybridoma clones 12D10, 2A4, 5B12. Clone 12D10 was generated by the Pierre laboratory (Schmidt et al., 2009), and is commercially available (Merck, catalog number: MABE343 ). We generated 2A4 and 5B12. 2A4 cells and supernatant are freely available to the scientific community from the Developmental Studies Hybridoma Bank http://dshb.biology.uiowa.edu/PMY-2A4
      2. Anti-ribosomal P antibody: human polyclonal autoimmune antiserum (from lupus patients) which recognizes three proteins of the 60S ribosomal subunit, RPLP0, P1 and P2 (Immunovision, catalog number: HPO-0100 )
      3. Anti-lysyl-tRNA synthetase (KRS) antibody: rabbit polyclonal serum which recognizes KRS enzyme (Abcam, catalog number: ab31532 )
    2. Secondary antibodies
      1. Donkey anti-mouse Alexa Fluor 488 (Jackson ImmunoResearch, catalog number: 715-545-150 )
      2. Donkey anti-rabbit Alexa Fluor 594 (Jackson ImmunoResearch, catalog number: 711-585-152 )
      3. Donkey anti-human Cy5 (Jackson ImmunoResearch, catalog number: 709-175-149 )
  6. Solutions (see Recipes)
    1. Anisomycin stock solution (1,000x)
    2. Cycloheximide (CHX) stock solution (1,000x)
    3. Emetine dihydrochloride stock solution (1,000x)
    4. Harringtonine stock solution (1,000x)
    5. PMY stock solution (1,000x)
    6. Sodium arsenite stock solution (1000x)
    7. Phosphate buffered saline (PBS)
    8. Growth medium
    9. Labeling medium
    10. Labeling control medium
    11. Extraction buffer
    12. Wash buffer
    13. 3% PFA
    14. Co-extraction/fixation buffer
    15. Staining buffer (SB)

Equipment

  1. Ice bucket
  2. 37 °C water bath (JULABO, model: TW20 )
  3. Class II laminar flow hood (FASTER, model: SafeFAST Elite )
  4. Microscopy ultrafine tweezers (Electron Microscopy Sciences, catalog number: 78522-7 )
  5. PIPETMAN 1-20 µl (Sartorius, model: Proline® Plus, catalog number: 728030 )
  6. PIPETMAN 20-200 µl (Sartorius, model: Proline® Plus, catalog number: 728060 )
  7. PIPETMAN 100-1,000 µl (Sartorius, model: Proline® Plus, catalog number: 728070 )
  8. Slide tray (Glaswarenfabrik Karl Hecht, Assistent, catalog number: 2700/10 )
  9. Cell Incubator (Heraeus, model: HERACell )
  10. Centrifuge (Eppendorf, model: 5415 R )
  11. Microwave
  12. Microscopy
    Laser scanning confocal microscope: Leica TCS SP5 (Leica Microsystems, model: Leica TCS SP5 ) with an HCX PL APO lambda blue 63.0x 1.40 oil UV objective. We used type FF immersion liquid (Cargille-Sacher Laboratories, catalog number: 16212 )
    Note: Other comparable systems may be used.

Software

  1. LAS AF V2.3.1 software (Leica)
  2. Imaris (Bitplane) and Huygens Essential Software for image deconvolution using the classical maximum likelihood estimation algorithm (V3.6, Scientific Volume Imaging BV, Hilversum, The Netherlands)
  3. ImageJ (NIH) https://imagej.net/Downloads

Procedure

In the following protocol, we have included controls needed to ensure the specificity of both labeling and staining steps: 3 ‘translation inhibitor controls’ and 1 ‘no PMY’ control. Furthermore, we describe a quadruple staining protocol that permits simultaneous visualization of nuclear translation sites (PMY), large subunit ribosomal proteins (RPLP0, RPLP1, RPLP2), a component of the multi-synthetase complex (lysyl-tRNA synthetase, KRS) and DNA (Hoechst 33258).
As emphasized in ‘Materials and Regents #3 Solutions’, either emetine or CHX can be used as elongation inhibitors to ‘trap’ PMY in translating ribosomes. However, emetine has proven to be more efficient for PMY labeling than CHX (David et al., 2012a). Furthermore, being irreversible, emetine, unlike CHX does not have to be maintained in all solutions throughout the procedure.
Two RPM methods have been developed: the original procedure (Procedure A) and a co-fixation/extraction procedure (Procedure B), easier and more adapted for non-adherent and primary cell cultures. Furthermore, labeling non-adherent cells necessitates an additional step (Procedure C).

  1. Procedure A (Original RPM protocol)
    Day 1
    1. Pre-warm growth medium (see Recipes) in 37 °C water bath. In a cell culture hood, distribute 4 ml of warm growth medium per well in 5 wells of a 6-well plate. In each well, carefully place up to five non-overlapping coverslips using sterile forceps.
      Notes:
      1. Backup plan. Coverslips are easily broken and ‘accidents’ frequently happen. Moreover, it’s always interesting to perform RPM with multiple Abs. For these reasons, we usually work with 4 coverslips per well.
      2. High quality forceps with extremely sharp tips are recommended to enable easy removal of the coverslips from wells. Practice is required to develop the knack of picking them up from 24-well plates without breaking them.
      3. Avoid coverslip overlapping. In order to be certain that coverslips will not move around in the well and overlap each other: (1) always add the medium first; (2) gently press down the coverslips down to remove any air bubbles between coverslip and the bottom of the well, gently slide the coverslips in order to create an adhesive force.
    2. Transfer 1 ml of growth medium containing 0.5 x 106 HeLa cells per ml in each well. Softly shake the plates to distribute cells uniformly in the wells. Incubate for 24 h to allow HeLa cells to attach tightly to the coverslips and spread. Immunofluorescent resolution is maximized when visualizing well spread cells.

    Day 2
    1. Examine cells using an inverted microscope to ensure they have reached 70-90% confluence. Several solutions must be prepared, at distinct temperatures.
      Warm to 37 °C:
      1. 10 ml of growth medium.
      2. 4 ml of freshly prepared labeling medium (prepared extemporaneously, see Recipes).
      3. 1 ml of labeling control medium (prepared extemporaneously, see Recipes).
      Chill on ice:
      1. 40 ml of PBS buffer (see Recipes).
      2. 5 ml of extraction buffer (freshly made, see Recipes).
      3. 5 ml of wash buffer (freshly made, see Recipes).
      Prepare 5 ml of 3% PFA (see Recipes) and maintain at RT.
    2. Optional step: Ensuring the specificity of PMY labeling necessitates pre-incubation with several translation inhibitors (see Table 1). This step is mandatory when working with new cell lines. The described protocol is designed to accommodate 3 ‘translation inhibitor controls’, preventing PMY labeling in different ways. Dilute each inhibitor in 2 ml of pre-warmed growth media. Assign a well for each: aspirate the media and replace it with media containing the corresponding inhibitor. Incubation times vary depending on the nature of the antibiotic and are indicated in Table 1.
      Note: Necessary controls. When performing RPM procedure for the first time (or with a new cell line), we recommend using at least 3 control conditions:
      1. Without ‘active ribosome’, using a translation initiation inhibitor (such as Harringtonine) that results in ribosome run off of mRNA, maximally releasing nascent chains.
      2. Without ‘ribosome catalyzed PMY incorporation’, i.e., no PMY.
      3. Using a PMY competitor, such as anisomycin, and in the ‘absence of antigen’, i.e., without PMY labeling.

        Table 1. Protein synthesis inhibitors


    3. Aspirate media in each well and replace with 900 µl of pre-warmed labeling medium (test well + 3 ‘inhibitor controls’ wells) or labeling control (in the last well). Incubate for 5 min at 37 °C.
      Note: Co-incubation with PMY and elongation inhibitor. Because emetine freezes translation instantly, it can be added simultaneously with PMY.
      Reversibility. Anisomycin, being a reversible competitor of PMY, we recommend adding anisomycin in the corresponding well during the PMY labeling incubation (to maintain a constant anisomycin concentration).
    4. Place the 6-well plate on ice, aspirate the medium and wash with 5 ml of ice-cold PBS.
    5. Aspirate PBS and add 1 ml of ice-cold extraction buffer in each well. Incubate for 2 min on ice.
      Note: Be careful with pipetting. It’s crucial from this step to slowly add buffer down the side of the well and avoid detaching the cells.
    6. Aspirate extraction buffer and extremely gently add 900 µl of ice-cold wash buffer.
    7. Aspirate gently on the side and extremely gently add 900 µl of freshly made 3% PFA. Incubate for 15 min at RT. Then, replace the fixing solution with 2 ml of ice-cold PBS. Check the wells using an inverted microscope to make sure that cells are still attached.
      Note: Storage prior to staining. Following PFA fixation, cells may be kept for at least 7 days at 4 °C (in PBS) without noticeably affecting the quality of the RPM staining. Likewise, stained coverslips can be stored for long periods (years, even decades) at -20 °C and retrieved.
    8. Proceed to ‘immunostaining procedure’ (Procedure D).

  2. Procedure B (co-extraction/fixation procedure)
    1. Follow Steps 1-6 of the ‘original RPM procedure’ (Procedure A).
    2. Aspirate PBS and add 1 ml of ice-cold Co-extraction/fixation buffer (see Recipes). Incubate for 20 min on ice.
    3. Aspirate Co-extraction/fixation buffer.
    4. Add 900 µl of freshly made 3% PFA. Incubate for 10 min at RT. Then, replace the fixing solution with 2 ml PBS.
    5. Proceed to ‘immunostaining procedure’ (Procedure D).

  3. PMY labeling procedure on non-adherent cells
    This procedure should be employed for non-adherent cells such as human peripheral blood monocytes (David et al., 2012b). It permits efficient attachment of cells to coverslips within minutes.
    1. ‘Alcian blue treated coverslips’ must be prepared in advance as followed:
      1. Prepare a solution of 1% (w/v) Alcian blue (Sigma-Aldrich) in distilled water. This solution lasts for months at RT.
      2. Completely cover 100-200 coverslips in few ml of this solution in a microwave-safe container.
      3. Stir manually, making sure that all coverslips are coated with Alcian blue (coverslips tend to stick together).
      4. Heat for 30 sec to 1 min in the microwave (maximum power intensity, the solution should boil for few seconds).
      5. Mix again manually, by shaking the container.
      6. Heat again until boiling.
      7. Discard Alcian blue solution.
      8. Wash with distilled water and with a gloved hand dissociate aggregated coverslips.
      9. Wash with 70% ethanol until only a very light blue shade remains on coverslips.
      10. Thoroughly wash with water.
      11. Separate coverslips by hand on a large piece of Whatman paper and let them dry.
      12. Alcian blue coated coverslips can be kept for months at RT.
    2. Place one Alcian blue treated coverslip per well of a 24-well plate.
    3. Centrifuge cells at 400 x g for 5 min at room temperature, wash cells by resuspending them with 5 ml warm DMEM twice in order to remove any trace of FBS.
      Note: Do not use FBS with Alcian blue coverslips. Alcian blue binds to negatively charged macromolecules such as glycosaminoglycans. The presence of FBS in the medium would inhibit Alcian blue association with membrane glycoproteins and prevent cell adhesion on coverslip.
    4. Resuspend cells in a small volume of DMEM (at least 106 cells/ml). Spot one drop of cells (about 50 µl) per coverslips.
    5. Incubate at 37 °C for 5 to 15 min (the more you wait, the better they stretch out).
    6. Aspirate the medium and replace it with 1 ml of ice-cold PBS.
    7. Proceed to the ‘Co-extraction/fixation procedure’ (Procedure B), starting with Step B7.

  4. Immunostaining procedure
    1. Transfer one coverslip of each condition into a 24-well plate (save the others at 4 °C). Then incubate cells with 500 µl of staining buffer (SB) (see Recipes) for 15 min at RT. Meanwhile, dilute primary antibodies in staining buffer: anti-PMY mAb (depending on the clone, final concentration varies between 1-4 µg/ml), anti-KRS Abs (1/200) and anti-ribosomal P Abs (1/5,000). To minimize non-specific binding of secondary antibodies, we usually supplement primary antibodies with 5% serum from the species used to generate the secondary antibodies (typically donkey antibodies from Jackson ImmunoResearch).
      Note: Staining buffer components. Glycine will quench the fixative properties of PFA. Saponin will facilitate the accessibility of the antibody to some epitopes. FBS can decrease non-specific binding of primary antibodies.
    2. Lay down a small piece of Parafilm. If needed, tape it on the bench. Spot 30 µl of diluted primary antibodies on Parafilm. This step is used to minimize the amount of primary Abs needed for staining. In 24-well plates, 200 µl is needed to completely cover the coverslip. Staining in 24-well plates will reduce the effort required and minimize errors. While antibodies can be reused, mAbs are generally available in essentially unlimited amounts if you have the hybridoma.
    3. Using forceps, carefully remove the coverslip, remove excess staining buffer by gently blotting coverslip edge on a Kimwipe and place cells side down on the primary antibody drop on Parafilm. Cover with a Petri dish with a moist paper towel attached to the inner top and incubate at room temperature for 60 min.
      Notes:
      1. Staining with ‘precious’ antibody. In order to limit the use of precious antibodies you can spot only 10 µl. In this case, you definitely need to incubate in a ‘moist chamber’ to prevent coverslips from drying out.
      2. When you inadvertently drop the coverslip. Inevitably, you will drop coverslips and will need to determine the ‘cell side’. This can be done if cells are sufficiently dense by holding up the light and looking for the side with a white film. If unsure, you can scratch the suspected side and see the loss of cells, or place the coverslip on an inverted microscope.
    4. Meanwhile, dilute secondary antibodies in staining buffer: 1/500 for goat anti-mouse A488, 1/500 for donkey anti-rabbit Alexa Fluor 594, 1/500 for donkey anti-human Cy5.
    5. With forceps, remove coverslips from primary antibody spots, and place in 24-wells plate. Wash three times with 1 ml 1x PBS.
    6. Lay down a small piece of Parafilm. If needed, tape it on the bench. Spot 30 µl of diluted secondary antibodies solution on Parafilm.
    7. Using forceps, carefully remove the coverslip from the plate, remove excess wash buffer by gently blotting coverslip edge on a Kimwipe and place cells side down on the secondary antibody drop on Parafilm, cover Petri dish with aluminum foil (to protect the fluorophore), incubate for 45 min at room temperature.
    8. With forceps, pick up coverslips from secondary antibody spots, and place them in a 24-well plate. Wash twice with 1 ml 1x PBS. Then wash again with 1 ml distilled water. Dilute Hoechst 33258 in distilled water (1 µg/ml).
      Note: PBS does not solubilize Hoechst 33285. For this reason, we recommend using distilled water for washes after this step.
    9. Aspirate distilled water and add 200 µl of diluted Hoechst solution. Incubate for 5 min at RT.
    10. Aspirate and wash twice with distilled water.
    11. Place a drop of Fluoromount-G (5 µl) on slides.
      Notes:
      1. Fluoromount-G. This mounting solution is quite viscous when cold. To facilitate the pipetting of small volumes (5 µl), we usually warm up the solution at RT for 15 min before using. Another helpful trick: cutting the tip of the plastic tip with a razor or scissor helps.
      2. The number of coverslips per slide. With practice, up to 8 coverslips can be placed on each slide. It is greatly advantageous to minimize the number of slides that have to be manipulated during microscopy which should be performed in the dark to accommodate the eyes and maximize visual acuity. Generally, the focal plane only has to be established one time for each slide, allowing rapid viewing of coverslips on the same slide. Give careful thought to the order of the coverslips on the slide. Put the most important coverslips for comparison with each other as closely as possible on the slide. The most important antibodies should be visualized with colors that can be seen by eye. It is important to form a general impression of staining of as many cells as possible, and this is by far most easily done by eye. Equally important is to write down your conclusions of the staining during or immediately after viewing the slides.
    12. Using forceps carefully pry up an edge and remove the coverslip, gently place cell side down on a Kimwipe to remove water and then place cells side down on mounting solution drops.
    13. Place slides in a tray and leave them dry overnight at room temperature in a drawer to protect the fluorophore.
      Note: Fast dry. If needed, drying may be hastened by incubating slides at 37 °C for 2-3 h. Never examine slides before mountant is dry, as this can damage the extremely expensive oil immersion objectives.
    14. Store the tray at 4 °C until analysis. Staining is stable for at least 2 weeks at 4 °C in darkness. For a longer storage we recommend using slide boxes and storing at -20 °C.
    15. Analyze with a confocal microscope (Figure 2).


      Figure 2. Deconvolved images of HeLa cell labeled with RPM (from David et al., 2011). HeLa cells were pulsed with PMY + CHX to label translating ribosomes and extracted with Dig to remove free PMY and cytosolic components. Cells were then fixed, permeabilized and stained for KRS, PMY or ribosomal P proteins. Co-localization was estimated using ImageJ (NIH) and JACoP plugin that compiles general co-localization indicators such as Pearson’s coefficient (Manders et al., 1992) and Van Steensel’s CCF (Van Steensel et al., 1996). KRS and RPM demonstrate extensive co-localization as quantitated by Van Steensel’s CCF greater than 0.75 and Pearson’s coefficient (R) greater than 0.5. Bar scales, 10 μm, 5 μm for Z1. Reproduced from David et al., 2011 with permission of the publisher and of Dr. Yewdell.

Data analysis

Images can be processed using LAS AF software (Leica), Imaris (Bitplane), Huygens Essentials Software (Version 3.6, Scientific Volume Imaging BV), Photoshop CS2 (Adobe), and/or ImageJ. For obvious ethical reasons, gamma function–which connects the numerical value of a pixel with its actual luminance–must not be manipulated. Each set of images for a given experiment must be processed identically to maintain the image intensity ratio. In previous publications (David et al., 2011; 2012a and 2012b; Macari et al., 2015), ImageJ and Prism software were used for quantitation and statistical analysis. Comparing translation activity from different condition necessitates acquisition of multiple fields (at least 6 fields per condition for statistical significance). In order to normalize each field, the mean fluorescence ratio of PMY/ribo P staining must be quantitated using ImageJ. Then, values may be plotted (mean ± SEM). For statistical analysis, we previously used two-tailed unpaired t-test. An example is presented in the following paper: David et al., 2012.

Notes

As stated above (methods #4), multiple controls are necessary when applying RPM for the first time or using new cell lines.

Recipes

  1. Anisomycin (Calbiochem) stock solution (1,000x)
    10 mg/ml (or 37 mM) in 100% ethanol solution
    Store at -20 °C
  2. Cycloheximide (CHX) stock solution (1,000x)
    100 mg/ml (or 355 mM) in 50% ethanol
    Store at -20 °C
  3. Emetine dihydrochloride stock solution (1,000x)
    25 mg/ml (or 45 mM) in 50% ethanol
    Store at -20 °C
  4. Harringtonine stock solution (1,000x)
    2 mg/ml (or 3.7 mM) in 100% ethanol
    Store at -20 °C
  5. PMY stock solution (1,000x)
    50 mg/ml (or 91 mM) in 50% ethanol
    Store at -20 °C
  6. Sodium arsenite stock solution (1000x)
    65 mg/ml (or 500 mM) in distilled water
    Store at 4 °C
  7. Phosphate buffered saline (PBS)
    210.0 mg/L KH2PO4
    9,000 mg/L NaCl
    726.0 mg/L Na2HPO4·7H2O
  8. Growth medium
    Dulbecco’s modified Eagle’s medium with:
    Glutamine 2 mM final
    1% penicillin/streptomycin
    7.5% fetal bovine serum (FBS)
  9. Labeling medium
    Add 5 µl of PMY stock solution (91 µM final) and either 5 µl of emetine stock solution (45 µM final) or 5 µl of CHX stock solution (355 µM final) to 5 ml growth medium
  10. Labeling control medium
    Add either 5 µl of emetine stock solution (45 µM final) or 5 µl of CHX stock solution (355 µM final) to 1 ml growth medium
  11. Extraction buffer
    0.015% (m/v) digitonin
    50 mM Tris-HCl pH 7.5
    5 mM MgCl2
    25 mM KCl
    355 µM CHX
    1x EDTA-free protease inhibitors (1 tablet per 10 ml)
    10 U/ml RNase Out
    DEPC treated water
  12. Wash buffer
    50 mM Tris-HCl pH 7.5
    5 mM MgCl2
    25 mM KCl
    355 µM CHX
    1x EDTA-free protease inhibitors (1 tablet per 10 ml)
    10 U/ml RNase Out
    DEPC treated water
  13. 3% PFA
    Dilute stock solution (16%) in 1x PBS
  14. Co-extraction/fixation buffer
    0.015% (m/v) digitonin
    50 mM Tris-HCl pH 7.5
    5 mM MgCl2
    25 mM KCl
    0.2 M sucrose
    355 µM CHX
    1x EDTA-free protease inhibitors (1 tablet per/10 ml)
    10 U/ml RNase Out
    3% PFA
    DEPC treated water
  15. Staining buffer (SB)
    0.05% saponin
    10 mM glycine
    5% FBS
    1x PBS

Acknowledgments

JWY is generously supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases. AD benefits from generous funding from Fondation pour la Recherche Médicale, Ligue contre le Cancer and Cancéropôle GSO. The authors declare no conflicts of interest or competing interests.

References

  1. Biever, A., Puighermanal, E., Nishi, A., David, A., Panciatici, C., Longueville, S., Xirodimas, D., Gangarossa, G., Meyuhas, O., Herve, D., Girault, J. A. and Valjent, E. (2015). PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons. J Neurosci 35(10): 4113-4130.
  2. David, A., Bennink, J. R. and Yewdell, J. W. (2012a). Emetine optimally facilitates nascent chain puromycylation and potentiates the ribopuromycylation method (RPM) applied to inert cells. Histochem Cell Biol 139(3): 501-504.
  3. David, A., Dolan, B. P., Hickman, H. D., Knowlton, J. J., Clavarino, G., Pierre, P., Bennink, J. R. and Yewdell, J. W. (2012b). Nuclear translation visualized by ribosome-bound nascent chain puromycylation. JCB 197(1): 45-57.
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简介

尽管氨基酸的同位素标记仍然是用于定量翻译速率的领域中的参考方法,但是其不提供关于翻译位点的空间定位的任何信息。 开发ribopuromycylation方法(RPM)的基本原理主要是在亚细胞水平上绘制翻译位点,同时避免检测从核糖体释放的新合成的蛋白质。 RPM通过使用嘌呤霉素特异性单克隆抗体的固定的和透化的细胞中的标准免疫荧光显微镜可视化主动翻译细胞中的核糖体以检测捕获在用链延长抑制剂处理的核糖体上的嘌呤化新生链。

【背景】几十年来,氨基酸的同位素标记被认为是研究蛋白质翻译的金标准。虽然这种方法已被证明是非常准确的评估翻译率,它没有提供翻译核糖体的位置信息。最近,氨基酸类似物使荧光检测新生链(Dieterich等人,2007)。然而,几乎所有检测到的信号都来自核糖体释放的多肽。我们最初的想法是开发一种方法来标记新生链,同时还束缚于翻译核糖体。

嘌呤霉素(PMY)是一种氨基糖苷类抗生素,模拟带电荷的tRNA Tyr并掺入核糖体A位点。因此,PMY通过核糖体催化 - 共价结合进入新生链COOH-末端(Pestka,1971),然后释放PMY-肽引发过早的翻译终止。最初产生PMY多克隆抗体(Abs)(Eggers等人,1997),以通过免疫印迹和免疫沉淀检测从核糖体释放的嘌呤化新生链。随后,荧光PMY被用于通过显微镜标记新生链(Starck等人,2004)。 Schmidt等(2009)发现,暴露于PMY的细胞产生足够量的PMY-终止的细胞表面蛋白,以使得能够使用单克隆Ab(mAb)通过活细胞流式细胞术进行检测,提供了一种测量的翻译率。重要的是,这些方法都不能区分核糖体连接的或释放的PMY-肽,并且在一定程度上被阻碍作为通过释放的蛋白质的降解进行翻译的量度。

已经发现链延长抑制剂,例如放线菌酮(CHX)或吐根碱,阻止PMY-新生链从核糖体中释放,我们开发了核糖核酸分析方法(RPM),使得能够在亚细胞水平上免疫荧光检测翻译位点(图1)(David等人,2011和2012b)。这种方法已被我们的实验室和许多其他实验室用于研究神经元中的翻译(Biever et al。,2015; Perry et al。,2016; ,2016),迁移细胞(Willett等人,2011),免疫细胞(Seedhom等人,2016)和受压或感染细胞(Kedersha等人,2016; Emmott等人,2017; Roth等人,2017)。

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图1. RPM的示意图(来自David等人,2012b)。用延伸抑制剂冷冻多核糖体(步骤1)后, ,将PMY加入(步骤2)到活细胞中,并通过核糖体催化使新生链成为嘌呤化(步骤3)。抗PMY单克隆抗体通过间接免疫荧光检测嘌呤化新生链(步骤4)。转载自David等人(2012b),得到出版商和Yewdell博士的许可。

关键字:翻译位点, 嘌呤霉素, Ribopuromycylation, 核糖体, 新生链

材料和试剂

  1. 物料
    1. 6孔和24孔板(Corning,Costar ,目录号:3506,3524)
    2. 高品质的玻璃盖玻片,直径12mm,#1厚度(Glaswarenfabrik Karl Hecht,Assistent,目录编号:1001/12)
      注意:使用前先用70%乙醇高压灭菌或灭菌。
    3. 显微镜载玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:J1800AMNZ)
    4. Whatman纸(GE Healthcare,目录号:3030-917)
    5. Parafilm(Bemis,产品目录号:701606)
    6. 培养皿(康宁,目录号:430591)
    7. 铝箔(Sigma-Aldrich,目录号:Z691569)
      制造商:希思罗科学公司,产品目录号:HD23534A。
    8. 塑料技巧
  2. 细胞系(S)
    所描述的程序和相关图(图2)使用HeLa细胞(ATCC,目录号:CCL-2.1)。
    注意:虽然这个程序最初是为HeLa细胞设计的,但它可以很容易地适用于其他贴壁细胞系(Graber et al。,2013)。然而,需要控制 来验证任何类型的调整(例如,对于非贴壁细胞,我们描述了一个替代方案(程序D)。
  3. 试剂
    1. 阿尔新蓝(Sigma-Aldrich,目录号:A5268)
    2. 70%乙醇(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R40135)
    3. Hoechst 33258(Thermo Fisher Scientific,Invitrogen TM,目录号:H1398)
    4. Fluoromount-G(SouthernBiotech,目录号:0100-01)
    5. 磷酸二氢钾(KH 2 PO 4)
    6. 氯化钠(NaCl)
    7. Na 2 HPO 4•7H 2 O
    8. Dulbecco's改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:41966029)
    9. 谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
    10. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:10378016)
    11. 胎牛血清(FBS)(Eurobio,目录号:CVFSVF0101)
    12. Digitonin(Wako Pure Chemical Industries,目录号:043-21376)
    13. Tris-HCl pH 7.5(Thermo Fisher Scientific,Invitrogen TM,目录号:15567027)
    14. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M0250)
    15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    16. 完整的无EDTA无蛋白酶抑制剂片剂(Sigma-Aldrich,Roche Diagnostics,目录号:11836170001)
    17. RNase Out(Life Technologies,目录号:100000840;或Thermo Fisher Scientific,Invitrogen TM,目录号:10777019)。
    18. DEPC处理的水(Thermo Fisher Scientific,Invitrogen TM,目录号:750023)
    19. 16%多聚甲醛(PFA)(电子显微镜科学,目录号:15710)
    20. 蔗糖(Sigma-Aldrich,目录号:84097)
    21. 皂苷(Sigma-Aldrich,目录号:84510)
    22. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  4. 蛋白质合成抑制剂(见表1)
    1. 茴香霉素(Sigma-Aldrich,目录号:A9789)
    2. 环己酰亚胺(CHX)(Sigma-Aldrich,目录号:C7698)
    3. 依地酸二盐酸盐(Sigma-Aldrich,目录号:E2375)
    4. 三尖杉酯碱(Santa Cruz Biotechnology,产品目录号:sc-204771)
    5. 嘌呤霉素(PMY)(Sigma-Aldrich,目录号:P7255)
    6. 亚砷酸钠(NaAsO 2)(Sigma-Aldrich,目录号:S7400)
  5. 抗体
    1. 一抗
      1. 抗PMY小鼠单克隆抗体:我们测试了来自不同杂交瘤克隆12D10,2A4,5B12的3个小鼠mAb。克隆12D10由Pierre实验室(Schmidt等人,2009)生成,并且可商购(Merck,目录号:MABE343)。我们生成了2A4和5B12。 2A4细胞和上清液可从发育研究杂交银行 http:// dshb。 biology.uiowa.edu/PMY-2A4
      2. 抗核糖体P抗体:识别60S核糖体亚基RPLP0,P1和P2(Immunovision,目录号:HPO-0100)的三种蛋白质的人多克隆自身免疫抗血清(来自狼疮患者)
      3. 抗赖氨酰-tRNA合成酶(KRS)抗体:识别KRS酶的多克隆兔血清(Abcam,目录号:ab31532)
    2. 二抗
      1. 驴抗小鼠Alexa Fluor 488(杰克逊免疫研究,目录号:715-545-150)
      2. 驴抗兔Alexa Fluor 594(Jackson ImmunoResearch,目录号:711-585-152)
      3. 驴抗人Cy5(Jackson ImmunoResearch,目录号:709-175-149)
  6. 解决方案(请参阅食谱)
    1. 茴香霉素储备液(1,000x)
    2. Cycloheximide(CHX)储备溶液(1,000x)
    3. 依米丁二盐酸盐储备液(1,000x)
    4. 三尖杉酯原液(1,000x)
    5. PMY库存解决方案(1,000x)
    6. 亚砷酸钠储备液(1000x)
    7. 磷酸盐缓冲盐水(PBS)
    8. 生长介质
    9. 标签介质
    10. 标签控制介质
    11. 提取缓冲区
    12. 清洗缓冲液
    13. 3%PFA
    14. 共同提取/固定缓冲液
    15. 染色缓冲液(SB)

设备

  1. 冰桶
  2. 37°C水浴(JULABO,型号:TW20)
  3. 二级层流罩(FASTER,型号:SafeFAST Elite)
  4. 显微镜超细镊子(电子显微镜科学,目录号:78522-7)
  5. PIPETMAN1-20μl(Sartorius,型号:Proline Plus Plus,目录号:728030)
  6. PIPETMAN20-200μl(Sartorius,型号:Proline Plus Plus,目录号:728060)
  7. PIPETMAN100-1,000μl(Sartorius,型号:Proline Plus,目录号:728070)
  8. 滑动托盘(Glaswarenfabrik Karl Hecht,Assistent,目录编号:2700/10)
  9. 细胞培养箱(Heraeus,型号:HERACell)
  10. 离心机(Eppendorf,型号:5415 R)
  11. 微波炉
  12. 显微镜
    激光扫描共聚焦显微镜:具有HCX PL APOλ兰色63.0×1.40油紫外物镜的Leica TCS SP5(Leica Microsystems,型号:Leica TCS SP5)。我们使用FF型浸渍液(Cargille-Sacher Laboratories,目录号:16212)
    注:可以使用其他类似的系统。

软件

  1. LAS AF V2.3.1软件(徕卡)
  2. Imaris(Bitplane)和惠更斯(Huygens)使用经典最大似然估计算法(V3.6,Scientific Volume Imaging BV,荷兰Hilversum)进行图像去卷积的基本软件
  3. ImageJ(NIH) https://imagej.net/Downloads

程序

在下面的协议中,我们已经包括确保标记和染色步骤的特异性所需的对照:3'翻译抑制剂对照'和1'不含PMY'对照。此外,我们描述了一个四重染色协议,允许同时可视化的核翻译网站(PMY),大型亚基核糖体蛋白(RPLP0,RPLP1,RPLP2),多合成酶复合物(赖氨酰tRNA合成酶,KRS)的一个组成部分和DNA (Hoechst 33258)。
正如“材料和试剂#3解决方案”中所强调的,依米丁或CHX可以用作延长抑制剂来“转移”核糖体中的PMY。然而,依米丁已被证明比CHX更有效地用于PMY标记(David等人,2012a)。此外,与CHX不同,不可逆转,依米丁不必在整个手术过程中保持所有的解决方案。
已经开发了两种RPM方法:原始程序(程序A)和共固定/提取程序(程序B),更容易且更适合于非贴壁和原代细胞培养。此外,标记非贴壁细胞需要额外的步骤(程序C)。

  1. 程序A(原始RPM协议)
    第1天
    1. 预温的生长培养基(见食谱)在37℃水浴中。在细胞培养罩中,在6孔板的5个孔中每孔分配4ml温生长培养基。在每个孔中,使用无菌镊子仔细放置五个不重叠的盖玻片。
      注意:
      1. 备份计划 封面容易被破坏,经常发生“意外”。而且,用多个Abs执行RPM总是很有趣的。由于这些原因,我们通常每口盖4个盖玻片。
      2. 建议使用尖端非常尖锐的高质量钳子,以便从盖玻片上轻松移除盖玻片。开发从24孔板中取出而不破坏它们的技巧是必需的。
      3. 避免盖玻片重叠。为了确保盖玻片不会在井中移动并相互重叠:(1)始终添加介质; (2)轻轻向下按下盖玻片以消除盖玻片和孔底部之间的气泡,轻轻地滑动盖玻片以产生粘合力。
    2. 在每个孔中转移1ml含有0.5×10 6 HeLa细胞/ ml的生长培养基。轻轻摇动平板,使细胞均匀分布在孔中。孵育24小时,让HeLa细胞紧紧地连接到盖玻片和传播。当可视化扩散细胞时,免疫荧光分辨率达到最大。

    第2天
    1. 使用倒置显微镜检查细胞,以确保它们达到70-90%汇合。必须在不同的温度下准备几种解决方案。
      暖至37°C:
      1. 10毫升的生长介质。
      2. 4毫升新鲜制备的标记介质(临时制备,见食谱)。
      3. 1ml标记对照介质(临时制备,参见食谱)。
      冰冷:
      1. 40毫升的PBS缓冲液(见食谱)。
      2. 5毫升提取缓冲液(新鲜制作,请参阅食谱)。
      3. 5ml洗涤缓冲液(新鲜制备,参见食谱)。
      准备5毫升的3%PFA(见食谱),并保持在RT。
    2. 可选步骤:确保PMY标记的特异性需要与几种翻译抑制剂预先孵育(见表1)。使用新的细胞系时,这一步是强制性的。所述的方案被设计为容纳3'翻译抑制剂对照,以不同方式防止PMY标记。将每种抑制剂稀释于2ml预热的生长培养基中。为每个分配一个孔:吸取介质并用含有相应抑制剂的介质替换。孵化时间根据抗生素的性质而有所不同,如表1所示。
      必要的控制 第一次执行RPM程序时(或使用新的单元格行),建议使用at至少3个控制条件:
      1. 没有“活性核糖体”,使用翻译起始抑制剂(如Harringtonine),导致核糖体流失mRNA,最大程度地释放新生链。
      2. 没有“核糖体催化的PMY掺入”,即没有PMY。
      3. 使用诸如茴香霉素的PMY竞争剂,并且在“不存在抗原”的情况下,即没有PMY标记。

        表1.蛋白质合成抑制剂


    3. 在每孔中吸取培养基,并用900μl预热的标记培养基(测试孔+ 3'抑制剂对照孔)或标记对照(在最后一个孔中) 。
      在37°C孵育5分钟 与PMY和延长抑制剂共同孵育 由于emetine立即冻结翻译,它可以与PMY同时添加。
      Anisomycin是PMY的可逆竞争者,我们建议在PMY标记孵育期间在相应的孔中加入茴香霉素(以维持恒定的茴香霉素浓度)。
    4. 将6孔板置于冰上,吸出培养基,并用5ml冰冷的PBS洗涤。
    5. 吸取PBS,并在每个孔中加入1ml冰冷的提取缓冲液。
      在冰上孵育2分钟 这一步至关重要,细胞。
    6. 抽取提取缓冲液,并轻轻地加入900μl冰冷的洗涤缓冲液。
    7. 轻轻地在一边吸取,并非常轻柔地添加900μL新鲜制成的3%PFA。在室温孵育15分钟。然后,用2毫升冰冷PBS替换定影液。用倒置显微镜检查孔,确保细胞仍然附着。
      注意: 在染色之前存放 。在PFA固定之后,细胞可以在4℃下(在PBS中)保持至少7天而不显着影响RPM染色的质量。同样,染色的盖玻片可以在-20°C长时间(几年,甚至几十年)储存和检索。
    8. 继续进行“免疫染色程序”(程序D)。

  2. 程序B(共同提取/固定程序)
    1. 遵循“原始RPM程序”(程序A)的步骤1-6。
    2. 吸出PBS并加入1毫升冰冷的共同提取/固定缓冲液(见食谱)。
      在冰上孵育20分钟
    3. 吸取共萃取/固定缓冲液。
    4. 加入900μl新鲜制成的3%PFA。在室温孵育10分钟。然后,用2毫升PBS代替定影液。
    5. 继续进行“免疫染色程序”(程序D)。

  3. 在非贴壁细胞上的PMY标记程序
    该程序应该用于非贴壁细胞如人外周血单核细胞(David等人,2012b)。它可以在几分钟内将细胞高效附着到盖玻片上。
    1. 必须事先准备“阿尔辛蓝处理过的盖玻片”
      1. 准备1%(W / V)阿尔新蓝(西格玛奥德里奇)蒸馏水的解决方案。
        这个解决方案在RT持续数月。
      2. 在微波安全的容器中,用几毫升的这种溶液完全覆盖100-200个盖玻片。
      3. 手动搅拌,确保所有的盖玻片都涂有阿尔新蓝(盖玻片往往粘在一起)。
      4. 在微波炉中加热30秒到1分钟(最大功率密度,溶液应煮沸几秒钟)。

      5. 手动摇动容器再次混合
      6. 再次加热直到沸腾。
      7. 放弃阿尔辛蓝解决方案。
      8. 用蒸馏水洗手,用戴手套的手将聚集的盖玻片分开。
      9. 用70%的乙醇清洗,直到盖玻片上只剩下很浅的蓝色。
      10. 彻底用清水洗净。

      11. 在一张大块的Whatman纸上用手分开盖玻片,让它们干燥。

      12. 阿尔辛蓝涂层的盖玻片可以保存几个月

    2. 每个孔放置一个阿尔辛蓝处理过的盖玻片
    3. 在室温下400×g离心细胞5分钟,通过用5ml温热的DMEM重悬细胞两次来洗涤细胞以除去任何微量的FBS。
      注意: 不要与阿尔辛蓝盖玻片一起使用FBS。 蓝色结合带负电荷的大分子,如糖胺聚糖。培养基中FBS的存在会抑制阿尔新蓝与膜糖蛋白的结合并阻止细胞在盖玻片上的粘附。
    4. 重悬细胞在一个小体积的DMEM(至少10 6细胞/毫升)。每盖玻片一滴细胞(约50μl)。

    5. 在37°C孵育5至15分钟(等待越多,伸展越好)
    6. 吸出培养基,并用1毫升冰冷的PBS代替。
    7. 从步骤B7开始,进行“共同提取/固定程序”(程序B)。

  4. 免疫染色程序
    1. 将每种条件的盖玻片转移到24孔板(4℃保存)。然后将细胞与500μl的染色缓冲液(SB)(参见配方)在室温孵育15分钟。同时,将稀释的第一抗体稀释于强染色缓冲液中:抗PMY单克隆抗体(取决于克隆,终浓度在1-4μg/ ml之间变化),抗KRS抗体(1/200)核糖体P抗体(1 / 5,000)。为了使二抗的非特异性结合最小化,我们通常用来自用于产生二抗(通常来自Jackson ImmunoResearch的驴抗体)的物种的5%血清补充一抗。
      注意: 染色缓冲液组分 甘氨酸会淬火PFA的固定特性。皂苷将促进抗体对一些表位的可及性。 FBS可以减少一级抗体的非特异性结合。
    2. 放下一小块Parafilm。如果需要的话,把它放在工作台上。在Parafilm上找到30μl稀释的一级抗体。这一步用于减少染色所需的主要抗体的数量。在24孔板中,需要200μl来完全覆盖盖玻片。在24孔板中染色将减少所需的工作量并最小化误差。虽然抗体可以重复使用,但是如果您有杂交瘤,单克隆抗体通常可以获得基本上无限的数量。
    3. 使用镊子,小心删除盖玻片,轻轻吸去盖玻片边缘上的Kimwipe,并将细胞面朝下的一级抗体滴在石蜡膜上,去除多余的染色缓冲液。盖上培养皿,用湿纸巾贴在内顶,在室温下孵育60分钟。
      注意:
      1. 使用“珍贵的”抗体进行染色。 为了限制使用贵重抗体,您只能检测到10μl。在这种情况下,您肯定需要在“潮湿的房间”孵化,以防止盖玻片干燥。
      2. 当你不小心掉下盖玻片时。 不可避免地,您将放弃盖玻片,并需要确定“细胞侧”。如果细胞足够密集,可以通过举起光线并用白色薄膜寻找一边进行。如果不确定,可以抓取疑似的一侧,看看细胞的损失,或将盖玻片放在倒置的显微镜上。
    4. 同时,稀释的第二抗体在强染色缓冲液中:羊抗鼠A488的1/500,驴抗兔Alexa Fluor 594的1/500,驴抗人Cy5的1/500。 />
    5. 用镊子,从主要抗体斑点去除盖玻片,并放置在24孔板。用1ml 1x PBS洗三次。
    6. 放下一小块Parafilm。如果需要的话,把它放在工作台上。在封口膜上找到30μl稀释的二抗。
    7. 使用镊子,小心地从平板上取下盖玻片,轻轻吸去盖玻片边缘上的Kimwipe,并将细胞侧面向下放在封口膜上的二次抗体滴,多余的清洗缓冲液,盖铝箔陪替氏培养皿(保护荧光),孵化在室温下45分钟。
    8. 用镊子,从二抗斑点拿起盖玻片,并放置在24孔板。用1毫升1x PBS洗两次。然后再用1毫升蒸馏水清洗。稀释Hoechst 33258蒸馏水(1微克/毫升)。
      PBS不溶解Hoechst 33285。 由于这个原因,我们推荐在这一步之后使用蒸馏水洗涤。 em>
    9. 吸取蒸馏水,并添加200μL稀释Hoechst解决方案。在室温孵育5分钟。
    10. 用蒸馏水洗两次。
    11. 在玻片上放一滴Fluoromount-G(5μl)。
      注意:
      1. Fluoromount-G 这种安装解决方案在冷时非常粘稠。为了促进小体积(5μl)的移液,我们通常在使用前将溶液在RT温热15分钟。另一个有用的技巧:用剃刀或剪刀切割塑料尖端的帮助。
      2. 每张幻灯片的盖玻片数量 练习时,每张幻灯片最多可放置8片盖玻片。最大限度地减少在显微镜过程中必须操作的载玻片的数量,这些载玻片应在黑暗中进行以适应眼睛并最大化视敏度。一般来说,每张幻灯片只需要建立一次焦平面,可以在同一张幻灯片上快速查看盖玻片。仔细考虑幻灯片上盖玻片的顺序。把最重要的盖玻片,尽可能在幻灯片上进行比较。最重要的抗体应该用肉眼可见的颜色进行可视化。形成尽可能多的细胞染色的总体印象是非常重要的,这是迄今为止最容易通过眼睛完成的。同样重要的是在观看幻灯片期间或之后立即写下染色的结论。
    12. 使用镊子小心地撬起一个边缘,并取出盖玻片,轻轻地放置在一个Kimwipe单元格面朝下去除水,然后将细胞面朝下安装溶液滴。
    13. 将玻片置于托盘中,在室温下置于抽屉中干燥过夜以保护荧光团。
      快速干燥 如果需要,可以通过在37℃孵育载玻片2-3小时来加速干燥。在安装剂干燥之前不要检查载玻片,因为这会损坏极其昂贵的油浸物镜。
    14. 将纸盘保存在4°C直到分析。染色在黑暗中4°C至少稳定2周。对于更长的存储空间,我们建议使用滑动盒并存放在-20°C。
    15. 用共焦显微镜分析(图2)。


      图2.用RPM标记的HeLa细胞的解卷图像(来自David et al。,2011年)。 HeLa细胞用PMY + CHX脉冲标记翻译核糖体并用Dig提取以除去游离的PMY和胞质成分。然后固定细胞,透化并染色KRS,PMY或核糖体P蛋白。使用ImageJ(NIH)和JACoP插件来估计共定位,所述插件编辑诸如Pearson系数(Manders等人,1992)和Van Steensel的CCF(Van Steensel等人,1996)。 KRS和RPM显示了Van Steensel的CCF大于0.75和Pearson系数(R)大于0.5的量化的广泛共定位。 Z1为10μm,5μm的棒状刻度。在出版商和Yewdell博士的许可下,从David et al。转载2011年。

数据分析

可以使用LAS AF软件(Leica),Imaris(位平面),Huygens Essentials软件(版本3.6,科学卷影像BV),Photoshop CS2(Adobe)和/或ImageJ处理图像。出于显而易见的道德原因,不得操纵将像素的数值与其实际亮度相关联的伽马函数。给定实验的每组图像必须进行相同处理以保持图像强度比。在之前的出版物(David等人,2011; 2012a和2012b; Macari等人,2015)中,使用ImageJ和Prism软件进行定量和统计分析。比较来自不同条件的翻译活动需要获取多个字段(每个条件至少6个字段以获得统计显着性)。为了使每个领域正常化,必须使用ImageJ定量PMY / ribo P染色的平均荧光比率。然后,可以绘制数值(平均值±SEM)。对于统计分析,我们以前使用双尾不成对t检验。以下文章介绍了一个例子: David ,2012

笔记

如上所述(方法#4),第一次应用RPM或使用新的细胞系时,需要多重控制。

食谱

  1. Anisomycin(Calbiochem)储备液(1,000x)
    在100%乙醇溶液中10mg / ml(或37mM)
    在-20°C储存
  2. Cycloheximide(CHX)储备溶液(1,000x)
    在50%乙醇中100mg / ml(或355mM)
    在-20°C储存
  3. 依米丁二盐酸盐储备液(1,000x)
    在50%乙醇中的25mg / ml(或45mM)
    在-20°C储存
  4. 三尖杉酯原液(1,000x)
    在100%乙醇中2mg / ml(或3.7mM)
    在-20°C储存
  5. PMY库存解决方案(1,000x)

    50毫克/毫升(或91毫米)的50%乙醇 在-20°C储存
  6. 亚砷酸钠储备液(1000x)
    65毫克/毫升(或500毫米)的蒸馏水
    在4°C储存
  7. 磷酸盐缓冲盐水(PBS)
    210.0mg / L KH 2 PO 4 4 9,000毫克/升NaCl
    726.0 mg / L Na 2 HPO 4•7H 2 O
  8. 生长介质
    达尔伯克的改良鹰的媒介与:
    谷氨酰胺2毫米最终
    1%青霉素/链霉素
    7.5%胎牛血清(FBS)
  9. 标签介质
    向5ml生长培养基中加入5μlPMY储备溶液(最终91μM)和5μl吐根碱储备溶液(终浓度45μM)或5μlCHX储备溶液(终浓度355μM) br />
  10. 标签控制介质

    添加5μl依米丁储备液(终浓度45μM)或5μlCHX储备液(终浓度355μM)至1ml生长培养基
  11. 提取缓冲区
    0.015%(m / v)毛地黄皂苷
    50mM Tris-HCl pH 7.5
    5mM MgCl 2 2/2 25 mM KCl
    355μMCHX
    1×EDTA无蛋白酶抑制剂(每10毫升1片)
    10 U / ml RNase Out
    DEPC处理过的水
  12. 清洗缓冲液
    50mM Tris-HCl pH 7.5
    5mM MgCl 2 2/2 25 mM KCl
    355μMCHX
    1×EDTA无蛋白酶抑制剂(每10毫升1片)
    10 U / ml RNase Out
    DEPC处理过的水
  13. 3%PFA
    在1x PBS中稀释原液(16%)
  14. 共同提取/固定缓冲液
    0.015%(m / v)毛地黄皂苷
    50mM Tris-HCl pH 7.5
    5mM MgCl 2 2/2 25 mM KCl
    0.2 M蔗糖
    355μMCHX
    1个不含EDTA的蛋白酶抑制剂(1片/ 10毫升)
    10 U / ml RNase Out
    3%PFA
    DEPC处理过的水
  15. 染色缓冲液(SB)
    0.05%皂素
    10 mM甘氨酸
    5%FBS
    1x PBS

致谢

JWY得到国立过敏和传染病研究所校内研究室的大力支持。 AD得益于Fondation pour la RechercheMédicale,Ligue contre le Cancer和CancéropôleGSO的慷慨资助。作者声明不存在利益冲突或利益冲突。

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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bastide, A., Yewdell, J. W. and David, A. (2018). The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level. Bio-protocol 8(1): e2669. DOI: 10.21769/BioProtoc.2669.
  2. David, A., Dolan, B. P., Hickman, H. D., Knowlton, J. J., Clavarino, G., Pierre, P., Bennink, J. R. and Yewdell, J. W. (2012b). Nuclear translation visualized by ribosome-bound nascent chain puromycylation. JCB 197(1): 45-57.
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