Jun 22, 2022
Whole Mount In Situ Hybridization in Zebrafish
- D R Hammond-Weinberger1
- 1Murray State University
- D R Hammond-Weinberger: Protocol based on Thisse et al., 1993
- Weinberger Lab
Protocol Citation: D R Hammond-Weinberger 2022. Whole Mount In Situ Hybridization in Zebrafish. protocols.io https://dx.doi.org/10.17504/protocols.io.b74vrqw6
Manuscript citation:
K. Dunn, A. Vashisht, and D.R. Hammond-Weinberger. Comparative in situ hybridization protocols in zebrafish. BioTechniques. doi: 10.2144/btn-2022-0038.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 23, 2022
Last Modified: June 22, 2022
Protocol Integer ID: 61301
Abstract
Whole mount in situ hybridization protocol optimized for single gene detection using chromogenic substrates NBT/BCIP in zebrafish (Danio rerio). Options are included for bleaching and permeabilization. This protocol beings with tissue preparation and ends with glycerol mounting for imaging. Probe synthesis is not included. This protocol has been used for embryos/larvae from 24 - 72 hpf.
Tissue Prep
Tissue Prep
Dechorionate embryos, if needed.
Fix embryos in 500 µL 4% paraformaldehyde for 02:00:00 at Room temperature or overnight at 4 °C .
2h
Wash in 1 mL 100% MeOH at Room temperature for 00:10:00 . (1/3)
10m
Wash in 1 mL 100% MeOH at Room temperature for 00:10:00 . (2/3)
10m
Wash in 1 mL 100% MeOH at Room temperature for 00:10:00 . (3/3)
10m
Store at -20 °C long-term (can be months or longer)
Day 1
Day 1
6h
6h
Wear gloves and treat surfaces for RNAses.
Note
All reagents should be nuclease-free. Use barrier pipet tips.
Rehydrate the embryos
Wash embryos in 0.5 mL 75% Methanol/25% PBTween, rocking, for 00:05:00 at Room temperature in 1.5 mL centrifuge tubes.
Note
PBTween is 1x PBS + 0.1% Tween20
Safety information
Methanol is hazardous waste. All liquids and contaminated materials must be collected and disposed of properly.
5m
Wash embryos in 0.5 mL 50% MeOH / 50% PBTween, rocking, for 00:05:00 at Room temperature
5m
Wash embryos in 0.5 mL 25% MeOH / 75% PBTween, rocking, for 00:05:00 at Room temperature
5m
Wash embryos in 0.5 mL PBTween, rocking, for 00:05:00 at Room temperature (1/3)
5m
Wash embryos in 0.5 mL PBTween, rocking, for 00:05:00 at Room temperature (2/3)
5m
Wash embryos in 0.5 mL PBTween, rocking, for 00:05:00 at Room temperature (3/3)
5m
Optional: Bleach embryos in0.5 mL freshly-made 3% H2O2 + 1.79 millimolar (mM) KOH for up to 00:05:00 . Leave the tube caps open and monitor bleaching.
5m
Rinse in 0.5 mL PBTween (1/2)
Rinse in 0.5 mL PBTween (2/2)
Permeabilize tissue. Option 1: proteinase K - proceed to step 6.1. Option 2: acetone - proceed directly to step 6.3.
Note
Timing of this step is critical.
Option 1: Digest with 1 mL 10 µg /mL Proteinase K in PBTween at Room temperature for 00:05:00 (24 hpf) or 00:20:00 (48 hpf) or 00:30:00 (72 hpf) . Proceed to step 6.2
Note
Time is variable by a few minutes depending on proteinase K stock
55m
Refix tissue in 0.5 mL 4% PFA, rocking, at Room temperature for 00:20:00 . Skip to step 6.4.
Safety information
Paraformaldehyde (PFA) is hazardous. All liquids and contaminated materials must be collected and disposed of properly.
20m
Option 2: Incubate in 1 mL 80% acetone/ 20% diH2O at Room temperature for 00:20:00 .
20m
Wash in 0.5 mL PBTween, rocking, at Room temperature for 00:05:00 (1/3)
5m
Wash in 0.5 mL PBTween, rocking, at Room temperature for 00:05:00 (2/3)
5m
Wash in 0.5 mL PBTween, rocking, at Room temperature for 00:05:00 (3/3)
5m
Separate embryos into designated tubes (example: sense vs. anti-sense tubes) if not done previously.
Incubate in 250 µL prehybe in hybe oven set to 65 °C , rocking, for at least 04:00:00
Safety information
Formamide is hazardous. Liquids and contaminated materials must be collected and disposed of properly.
4h
Incubate with (0.1-1μg/mL) probe diluted in 250 µL warmed prehybe Overnight , 65 °C , rocking.
Note
Prehybe Recipe (10 mL ):
Mix together: 5 mL formamide, 1.5 mL 20x SSC, 50 µL 20% Tween20, 185 µL 0.5 Molarity (M) Citric acid, 10 µL heparin, 500 µL 10 mg / mL tRNA, and 2.75 mL nuclease-free water
OPTIONAL: mix in 0.5 g dextran sulfate
Day 2
Day 2
1h 40m
1h 40m
Remove probes. Probes can be stored at -20 °C and reused up to 3 times.
Post-hybridization washes
Wash in 0.5 mL 100 % (50% 5x SSC / 50% formamide) for 00:10:00 at 75 °C rocking
10m
Wash in 0.5 mL 75% (50% 5x SSC / 50% formamide) / 25% 2x SSC for 00:10:00 at 75 °C rocking
10m
Wash in 0.5 mL 50% (50% 5x SSC / 50% formamide) / 50% 2x SSC for 00:10:00 at 75 °C rocking
10m
Wash in 0.5 mL 25% (50% 5x SSC / 50% formamide) / 75% 2x SSC for 00:10:00 at 75 °C rocking
10m
Wash in 0.5 mL 2x SSC for 00:10:00 at 75 °C rocking
10m
Wash in 0.5 mL 0.2x SSC for 00:15:00 at 75 °C rocking
15m
Wash in 0.5 mL 0.2x SSC for 00:15:00 at 75 °C rocking
15m
Wash in 0.5 mL 75% 0.2x SSC / 25% PBTween for 00:10:00 at Room temperature rocking
10m
Wash in 0.5 mL 50% 0.2x SSC / 50% PBTween for 00:10:00 at Room temperature rocking
10m
Wash in 0.5 mL 25% 0.2x SSC / 75% PBTween for 00:10:00 at Room temperature rocking
10m
Wash in 0.5 mL 100% PBTween for 00:10:00 at Room temperature rocking
10m
Incubate in 0.5 mL block for at least 02:00:00 Room temperature , rocking
Note
Block solution: is 5% sheep serum, 2mg/mL BSA, and 1% DMSO in PBTween
For 10 mL : Mix 500 µL normal sheep serum, 0.2 g BSA, 100 µL DMSO, and 9.4 mL PBTween
2h
Incubate Overnight 4 °C with 0.5 mL 1:5000 sheep AP-conjugated anti-DIG Fab fragments (or 1:2000 sheep AP-conjugated anti-FLU Fab fragments)
4h
Day 3
Day 3
2h 30m
2h 30m
Remove antibody. Antibody can be stored at 4 °C and reused up to 3 times.
Post-antibody washes
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (1/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (2/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (3/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (4/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (5/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (6/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (7/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (8/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (9/10)
10m
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (10/10)
10m
Make fresh NTMT buffer. Mix 1 mL 1 Molarity (M) Tris 9.5 , 200 µL 5 Molarity (M) NaCl, 500 µL 1 Molarity (M) MgCl2, 50 µL Tween20 and 8.25 mL water
Equilibrate embryos in 0.5 mL NTMT buffer for 00:05:00 at Room temperature (1/2)
5m
Equilibrate embryos in 0.5 mL NTMT buffer for 00:05:00 at Room temperature (2/2)
5m
Transfer embryos to multiwell culture plate (keep the tubes)
Wash in 1 mL NTMT buffer for 00:05:00 at Room temperature
5m
Prepare fresh stain solution.
Add 4.5 µL /mL NBT and 3.5 µL / mL BCIP to NTMT buffer. Protect from light.
Safety information
NBT and BCIP are hazardous. Liquids and contaminated materials must be collected and disposed of properly.
Replace NTMT in culture plates with 1 mL of the freshly prepared NBT/BCIP stain solution.
Cover with foil
Stain in the dark until staining reaches desired intensity, typically when color begins to appear in the sense controls. This step can last hours to days.
Stop the reaction by rinsing in 2 mL PBTween
Fix tissue after staining
Transfer embryos back to their tubes
Fix embryos in 0.5 mL 4% PFA, rocking, at Room temperature for 00:20:00
20m
Wash in 0.5 mL PBTween, rocking, at Room temperature for 00:05:00 (1/3)
5m
Wash in 0.5 mL PBTween, rocking, at Room temperature for 00:05:00 (2/3)
5m
Wash in 0.5 mL PBTween, rocking, at Room temperature for 00:05:00 (3/3)
5m
Prepare embryos for glycerol imaging
Wash embryos in 1 mL 30% glycerol / 70% PBTween at Room temperature for 00:10:00 while rocking and covered in foil.
10m
Wash embryos in 1 mL 50% glycerol / 50% PBTween at Room temperature for 00:10:00 while rocking and covered in foil.
10m
Wash embryos in 1 mL 80% glycerol / 20% PBTween at Room temperature for 00:10:00 while rocking and covered in foil.
10m