RNA IN SITU HYBRIDIZATION AND IMMUNOFLUORESCENCE PROTOCOLS FOR MOUSE EMBRYOS

Mona B*, Uruena A*, Ma, Z, Borromeo MD, Kollipara RK, Chang JC, and Johnson JE. (2017) Repression by PRDM13 is critical for generating precise neuronal identity. eLIFE 6. pii: e25787. doi: 10.7554/eLife.25787. PMCID: PMC5576485

Prepared by Juan Villarreal (Juan.Villarreal@utsouthwestern.edu) and Trisha Savage (Trisha.Savage@utsouthwestern.edu) April 27, 2020

I. RNA IN SITU HYBRIDIZATION PROTOCOL FOR MOUSE EMBRYOS

Fixation/Embedding Tissues:

1. Remove      embryos from amniotic membranes in      cold PBS

2. Fix      in fresh cold 4%      paraformaldehyde for 2-3 hours at 4C for E9.5-E14.5 embryos, overnight for      embryos older than E14.5. Older embryos can be eviscerated before fixing to      increase access to the fixative if all tissues are not needed.

3. Rinse      3X in PBS at 4C for 10 min.

4. Sink      in 30% sucrose in PBS overnight at 4C.

5. Embed      in OCT and freeze on dry ice. (can store blocks at -80C until ready to      section)

6. Section      at 30µm on cryostat, keeping slides on slide warmer until finished.

7. Store      slides at -20C until ready to use.

3 Day In Situ Hybridization protocol

Day#1: Pre-Treatment and Hybridization of Sections

Note:              All reagents and slide mailers in the following steps must be RNAse-free!

1.     Bring slides to room temperature (RT) and then dry at 50°C for 15 minutes on slide warmer.

2.     Fix in 4% paraformaldehyde in DEPC-PBS at RT for 20 minutes in a trough.

3.     Wash 2x in DEPC-PBS at RT for 5 min Quick Wash

4.     Treat slides with 5µg/ml Proteinase K in PK buffer @ RT for 8-15min depending on age. 8.5min for E11.5 and younger, 9-11 min for E12.5 and older. New batches of PK may need to be recalibrated.

5.     Wash 1x in DEPC-PBS at RT for 5 min.

6.     Postfix in 4% paraformaldehyde in DEPC-PBS at RT for 20 minutes.

7.     Rinse once in DEPC-H₂O at RT by quickly dipping.

8.     To reduce non-specific binding, treat slides 10 minutes in a slowly stirring mixture of triethanolamine-HCL + acetic anhydride. (Mix 200ml 0.1M RNAse-free triethanolamine-HCl pH 8.0 plus 500 µl acetic anhydride and stir until beading disappears.)

9.     Wash 1x in DEPC-PBS at RT for 5 min.

10.  Prehybridize for 1-4 hours at 60-70°C in hybridization buffer in slide mailers.

11.  Hybridization with 1-2 ng/µl of probe in fresh (not used for prehybridization) heated hybridization buffer and continue incubation for a further 12-16 hours or O/N. (Prepare 1-2 ng/µl of RNA probe in 100µl Hybridization solution for each slide. Depending on the quality of the probe, the concentration can be adjusted as needed.)

Day#2: Washing steps and antibody binding

1.     Wash slides in a Copeland Jar or a glass trough with 2xSSC 60-65°C for 15 min.

2.     Wash once more in 2xSSC at RT for 5 min.

3.     RNAse treat with 10 µg/ml RNAse A and 1 U/ml RNase T1 in 2xSSC at 37°C for 30-45 min.

4.     Wash 2x in 2xSSC at RT for 5 min each.

5.     Wash 2x in high stringency 0.2xSSC at 60-65°C for 15-30 min each.

6.     Wash 1x in 0.2xSSC at RT for 2 min to equilibrate temperature.

7.     Wash 2x in PBT at RT for 20 min each.

8.     Block in PBT+10% inactivated sheep serum (SS) for 30 min – 1hr at RT in an antibody humidity chamber.

9.     Incubate O/N at 4°C or 1 hr at RT in 200-300 µL of anti-digoxygenin antibody (use dilution as per manufacturer’s instructions, dilute in PBT+10% inactivated sheep serum).

Day#3: Antibody Visualization of Digoxygenin

1.     Wash 3 times in PBT at RT for 20-30 min each in a trough.

2.     Wash 2 times in Alkaline Phosphatase buffer at RT for 5 min each.

3.     React with substrates NBT (Sigma 11383213001) and BCIP (Sigma 11383221001) 2-20 hours in the dark in a slide mailer. For every ml of Alkaline Phosphatase buffer, add 1 µl of NBT and 3.5µl of BCIP. Development time will depend on the abundance of the RNA.

4.     Wash 2 times in paper filtered PBS at RT. Mount the slides.

Troubleshooting and Tips

·Be extremely rigorous about how long your slides are in 0.2x SSC. High-stringency washes strip probe off sections. When using probes with low background, you can pare these washes back to improve signal.

·Check for particulates in the phosphate buffer used in making the fixative or sucrose for treating embryos.  Contamination for either reagent causes excessive background for antibody staining and severely reduces in situ signals.

If you rinse and reuse your mailers for NBT/BCIP development, sometimes precipitate forms in the mailer and is not rinsed out. Such precipitates can increase background—so rinse well.

·PK times can vary for different tissues and ages. It is important to optimize the amount of time the slides are in PK. Too little time and the probe cannot penetrate the tissue. Too long and it will digest the tissue leaving unviable samples. 

Probe Preparation

1.     Cut 10 µg of plasmid DNA with the appropriate enzyme, typically 1 U/µg for one hour.

2.     Purify with Qiagen PCR clean up kit, elute in 40 µL of nuclease-free H2O and store at –20°C until required. Check for complete digestion (0.5 µL) on a mini-gel.

Important note: Even very small amounts of DEPC can inactivate RNA polymerase. I have noted reductions in yield if I use DEPC treated water to make RNAse-free TE, even though the water was autoclaved before making TE. In the Johnson Lab protocol, don’t use TE – just use nuclease-free H2O.

3.     Set up the following reaction in 50 µL total volume: *

10x Ambion synthesis buffer (8151G)                        5 µL

10 mM dNTP’s/digoxygenin-UTP                       3 µL

Linearized DNA template                                  10 µL (2.5 µg)

RNA polymerase PLUS (T3, T7, SP6)   5 µL (100 units)

RNAse-free water to 50 µL                             27 µL

Incubate at 37°C for 2 hours.  Note: Add digoxygenin-UTP last.

*Depends on which kit you use, so best to follow manufacturer’s directions.

4.     Add 2 units of RNAse-free DNAse (1 µL of Ambion TURBO DNAse (2U/µL)) and continue incubation for 30 min at 37°C.  Remove 0.5 µL and run on 1% TAE mini gel to check that DNA has been degraded.  Any smearing below predicted band size indicates degradation.  Do not use probe if this occurs. 

5.     Separate unincorporated ribonucleotides using a Roche Quick Spin (TE) column (either G-25 or G-50 Sephadex).  Follow manufacturer’s directions.

6.     Run 0.5 µL on a clean, but not DEPC gel to check concentration and purity.  You can also quantitate at 260 nm if you wish, but it is not necessary. We determine concentration with nanodrop. Run an RNA denaturing gel if necessary, to get proper probe size.

7.     Store probe at -20^oC. Depending on the quality of the probe they can last up to 2 yrs, however, others only last a couple months. If it as been a while since the probe was used, run a few µl on a gel to check for degradation.

SOLUTIONS

RNase Free Solutions!!!

4% PFA (paraformaldehyde)

-Heat up 1X PBS in microwave, 30 sec for every 100 ml. Add 4g PFA powder per 100ml 1XPBS, stir in fume hood at RT and add NaOH to 0.1% by adding 10N NaOH (100µl 10N NaOH per 100ml) to help dissolve. Once all the PFA is dissolved add equal volume of 10N HCl (100µl 10N HCL per 100ml) in order to neutralize. Filter and store in 4°C for up to a week.

DEPC-PBS (1L)

-Filter 1L of 1XPBS into glass bottle, add 1ml of DEPC and stir for at least 10 min. Autoclave with 15 min Liquid cycle

DEPC-H₂O (1L)

-Filter 1L of MilliQ-H2O into glass bottle, add 1ml of DEPC and stir for at least 10 min. Autoclave with 15 min Liquid cycle

PK Buffer (1L)

-Add 6.057g of Tris Base to 10ml of 0.5M EDTA and QS to 1L with DEPC-H₂O.

PK Stock

            Proteinase K, recombinant, PCR Grade (Sigma 03115879001)

            Make 10mg/ml stock solution in ultra pure H2O. Store at 4^oC.

1M (10X) triethanolamine-HCL (500ml) (pH 8.0)

-Add 66.5 ml Triethanolamine and 20 ml concentrated HCL to 413.5 ml of DEPC-H₂O.

Hybridization Solution (50ml)

            -25ml of Formamide (deionized)

            -12.5ml of 20X SSC (DEPC-treated)

            -15mg of Yeast tRNA powder (Sigma 83853)

            -5mg of Heparin powder (Sigma-H3393-50KU)

            -500µl of 100x Denhardt’s Solution

            -500µl of 10%Tween-20/DEPC-H₂O

            -500µl of 0.5M EDTA pH8.0

            QS to 50ml with DEPC-H₂O

            *Make 500µl aliquots and store -20^oC freezer

Non-RNase Free Solutions            

PBT (1L)

            -Add 2g of BSA and 1ml of 100% Triton-X100 to 1L of filtered 1XPBS

Alkaline Phosphate Buffer (500ml)

            -50ml of 1M Tris-Cl (pH 9.5)

            -25ml of 1M MgCl₂

            -10ml of 5M NaCl

            QS to 500ml with ddH₂O

20X SSC pH 7.0 (1L) *(if for Hybridization Solution use DEPC-H₂O) 

            -175.3g of NaCl

            -88.2g of Sodium Citrate

            QS to 1L with ddH₂O

10X PBS (1L) (pH 6.8) 

            -80g of NaCl

            -2g of KCl

            -2g of KH₂PO₄

-21.6g of Na₂HPO₄ ● 7H₂O

QS to 1L with ddH₂O and stir overnight

            *For 1X PBS add 100ml of 10X PBS to 900ml of ddH₂O (pH 7.4)

II. Standard Protocol for ImmunoFLUORESCENCE oN Mouse/Chick embryo sections

Fixation/Embedding Tissues:

1. Remove      embryos from amniotic membranes in      cold PBS

2. Fix      in fresh cold 4%      paraformaldehyde for 2-3 hours at 4C for E9.5-E14.5 embryos, overnight for      embryos older than E14.5. Older embryos can be eviscerated before fixing      to increase access to the fixative if all tissues are not needed.

3. Rinse      3X in PBS at 4C for 10 min.

4. Sink      in 30% sucrose in PBS overnight at 4C.

5. Embed      in OCT and freeze on dry ice. (can store blocks at -80C until ready to      section)

6. Section      at 30µm on cryostat, keeping slides on slide warmer until finished.

7. Store      slides at -20C until ready to use.

Immunostaining of tissue sections:

Note: It is helpful to do all procedures in the dark as much as possible to avoid photobleaching for fluorescent antibodies. It is also important not to let a slide dry out after the sections have been rehydrated.

1.         Take slides out of -20^oC and dry at room temperature for no more than 10 minutes. Label slide with antibodies to be applied.

2.         Rehydrate the slides by washing in PBS for 10 minutes three times. Use trough and/or slide holder.

3.         One at a time, take slides out of PBS. Dry back of slide and frosted area. Using suction, dry around sections, but not sections directly. Use a Pap pen to place a water barrier around the sections.

4.         Place slide flat in humid chamber and add Block solution to cover all sections. If using several slides, blocking can be done in a slide mailer with Block solution.  Block solution can be saved and reused at least 5-6 times if kept at 4^oC in between use.

5.         After Block has been added to all slides or the slides have been put in mailer, close humidity chamber (or slide mailer) and allow slides to block at RT 1 hour. 

6.         Dilute primary antibodies to appropriate concentrations using Block solution. This is the primary antibody solution. Antibodies generated in different species can be mixed for double and triple labeling.

7.         One at a time, suction off Block solution from a slide, and then add antibody solution (100-300 µl or enough to cover the sections) and place in humidity chamber. After one slide has antibody solution applied, move to next slide. Repeat for all slides and avoid drying out of sections.

8.         Once all slides have antibody solution applied, place lid on humidity chamber and gently move to a flat area in the 4^oC refrigerator. Incubate overnight, approx. 16 hours.

9.         After incubation, remove humidity chamber from refrigerator. Remove antibody solution from each slide (one at a time) using a pipette to save the primary antibody solution.  (Primary antibodies are precious, so keep and reuse. (Store at 4^oC, reuse up to 2-3 times. Note, sometimes background improves with antibodies after multiple uses.) Place slide in PBS to wash for 10 minutes three times. Use trough and slide holder.

10.      Dilute fluorescently labeled secondary antibodies in PBS (as per manufacturer suggested dilution), making sure you use the correct secondary antibodies that will recognize your primary antibodies.

11.      One at a time, take slides out of PBS wash. Dry back of slide and frosted area.  Using suction, dry around sections, but not sections directly. Place in humidity chamber and add secondary antibody solution (100-300 µl or enough to cover the sections).

12.      After last slide has secondary antibody solution, close humidity chamber and incubate for one hour at room temperature. Over incubation may lead to high background.

13.      Remove secondary antibody solution from each slide (one at a time), no need to save.  Place slide in PBS to wash.

14.      Wash slides in PBS for 10 minutes three times. Use trough and slide holder.

15.      One at a time, take slides out of PBS. Dry back of slide and frosted area. Using suction, dry around sections, but not sections directly. Add 1-2 drops of mounting solution of your choice (Vectashield, H-1000-10, Prolong Diamond, P36970) and place appropriately sized coverslip. 

16.      Image on appropriate microscope for fluorescence detection.

17.      Store slides at 4^oC. Seal with fingernail polish if you want to store them for an extended time and did not use a hardening mounting solution.

Troubleshooting:

Background too high: increase dilution of primary and/or secondary antibodies and/or make new block solution.

No signal: make sure you have used the correct species-specific secondary antibody, decrease dilution of primary antibody, or adjust tissue fixation times.

SOLUTIONS:

10X PBS (1L) (pH 6.8)  

            -80g of NaCl

            -2g of KCl 

            -2g of KH₂PO₄

-21.6g of Na₂HPO₄ ● 7H₂O

QS to 1L with ddH₂O and stir overnight 

            *For 1X PBS add 100ml of 10X PBS to 900ml of ddH₂O (pH 7.4) 

4% Paraformaldehyde

Heat up PBS in microwave, 30 sec for every 100 ml. Add 4g PFA powder per 100 ml PBS, stir in fume hood at RT and add NaOH to 0.1% by adding 10N NaOH (100 µl 10N NaOH per 100 ml) to help dissolve. Once all the PFA is dissolved add equal volume of 10N HCl (100µl 10N HCL per 100ml) in order to neutralize. Filter and store at 4^oC for up to a week.

Block Solution

            1% Goat Serum

            0.1% NP-40 (Alternative: 0.1% Triton-X100 can be substituted but is a stronger     detergent)

            PBS

            Store at 4^oC, good for 6 months