Sep 06, 2022
Double Whole Mount In Situ Hybridization in Zebrafish
- D R. Hammond-Weinberger1
- 1Murray State University
- Weinberger Lab
Protocol Citation: D R. Hammond-Weinberger 2022. Double Whole Mount In Situ Hybridization in Zebrafish. protocols.io https://dx.doi.org/10.17504/protocols.io.b75frq3n
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 24, 2022
Last Modified: September 06, 2022
Protocol Integer ID: 61319
Keywords: chromogenic substrates in embryonic zebrafish, hpf zebrafish embryo, embryonic zebrafish, zebrafish this protocol, zebrafish, double whole mount in situ hybridization, chromogenic substrate, situ hybridization, whole mount in situ hybridization, several stain pairing, embryo, double whole mount, tissue preparation
Funders Acknowledgements:
NIGMS
Grant ID: 8P20GM103436
McNair Grant
Grant ID: #P217A090330
Abstract
This protocol has been optimized for serial detection of two chromogenic substrates in embryonic zebrafish (Danio rerio). Several stain pairings are included as options. Protocol begins with tissue preparation and ends with a glycerol series in preparation for imaging. This protocol has been successfully used on 24 hpf zebrafish embryos.
Protocol materials
Pierce™ DAB Substrate KitThermo FisherCatalog #34002
Troubleshooting
Tissue Prep
3h
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
5h
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 in 0.5 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 in0.5 mL PBTween (1/2)
Rinse in0.5 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 permeabilization 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)
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
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
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
5h
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
Note
Can sit overnight in this step
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)
Note
Staining with DAB requires the use of a peroxidase-conjugated enzyme, such as 1:200 POD-FLU.
4h
Day 3
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)
Note
Can sit overnight in this step
If staining with DAB, skip directly to step 13
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. Choose one of the following:
NBT/BCIP - Indigo stain: Add 4.5 µL /mL NBT and 3.5 µL / mL BCIP to NTMT buffer. Protect from light. Jump to step 16.
FastRed - Red stain - Dissolve buffer tablet(s) in 1 mL /tablet /tablet dH2O and sonicate 00:05:00 . Dissolve FastRed tablet(s) in buffer and sonicate for 00:05:00 . Jump to step 16.
10m
DAB - brown stain - Diaminebenzidine requires a peroxidase-conjugated antibody. Prepare 1x Pierce™ DAB Substrate KitThermo FisherCatalog #34002 in DAB buffer.
FR/BCIP - cyan stain - Dissolve buffer tablet(s) in 1 mL /tablet dH2O and sonicate 00:05:00 . Dissolve FastRed tablet(s) in buffer and sonicate for 00:05:00 . Add 3.5 µL /mL BCIP and 5.6 µL /mL FastRed to fresh NTMT. Vortex and let sit upright for 00:05:00
15m
Replace NTMT in culture plates with 1.5 mL of the freshly prepared 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.
Day 4
5h
Transfer embryos back to tubes. Protect from light in this and all subsequent steps.
Incubate 00:30:00 , rocking, in 0.1 Molarity (M) glycine HCl 2.2 plus 0.1% Tween at Room temperature to remove first antibody.
30m
Wash away glycine
Wash in 0.5 mL PBTween for 00:05:00 at Room temperature , rocking (1/4)
5m
Wash in 0.5 mL PBTween for 00:05:00 at Room temperature , rocking (2/4)
5m
Wash in 0.5 mL PBTween for 00:05:00 at Room temperature , rocking (3/4)
5m
Wash in 0.5 mL PBTween for 00:05:00 at Room temperature , rocking 4/4)
Note
Can sit overnight in this step
5m
Incubate embryos in 100 µL preabsorbed sheep AP-conjugated anti-DIG Fab fragments at a 1:2000 dilution in block. You can reuse the antibody 3x. Rock for 02:00:00 Room temperature or Overnight 4 °C .
Note
Staining with DAB requires the use of a peroxidase-conjugated enzyme, such as 1:200 POD-FLU.
4h
Day 5
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)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (2/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (3/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (4/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (5/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (6/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (7/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (8/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (9/10)
Wash in 0.5 mL PBTween for 00:10:00 at Room temperature , rocking (10/10)
Note
Can sit overnight in this step
If staining with DAB, skip directly to step 24
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)
Equilibrate embryos in 0.5 mL NTMT buffer for 00:05:00 at Room temperature (2/2)
Transfer embryos to multiwell culture plate (keep the tubes)
Wash in 1 mL NTMT buffer for 00:05:00 at Room temperature
Prepare fresh stain solution. Choose one of the following:
Note
Stain colors for first and second sequence must be compatible.
NBT/BCIP - Indigo stain: Add 4.5 µL /mL NBT and 3.5 µL / mL BCIP to NTMT buffer. Protect from light. Jump to step 27.
FastRed - Red stain - Dissolve buffer tablet(s) in 1 mL /tablet /tablet dH2O and sonicate 00:05:00 . Dissolve FastRed tablet(s) in buffer and sonicate for 00:05:00 . Jump to step 27.
10m
VectorRed - Red/yellow stain - To 5 mL of 0.1 Molarity (M) Tris-HCl 8.2 + 0.1% Tween, add 2 drops each of reagents 1, 2, and 3 of Vector Red Substrate kit. Mix well. Jump to step 27.
Note
VectorRed cannot be used as the first stain, per vendor instructions.
DAB - brown stain - Diaminebenzidine requires a peroxidase-conjugated antibody. Prepare 1x Pierce™ DAB Substrate KitThermo FisherCatalog #34002 in DAB buffer. Jump to step 27.
FR/BCIP - cyan stain - Dissolve buffer tablet(s) in 1 mL /tablet dH2O and sonicate 00:05:00 . Dissolve FastRed tablet(s) in buffer and sonicate for 00:05:00 . Add 3.5 µL /mL BCIP and 5.6 µL /mL FastRed to fresh NTMT. Vortex and let sit upright for 00:05:00 . Jump to step 27.
Citation
15m
Replace NTMT in culture plates with 1.5 mL of the freshly prepared 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.
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
Citations
Step 29.5
Hurtado R, Mikawa T. Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.