Double Whole Mount In Situ Hybridization in Zebrafish
D R. Hammond-Weinberger
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.
Steps
Tissue Prep
Dechorionate embryos, if needed.
Fix embryos in 500µL4% paraformaldehyde for 2h 0m 0s at Room temperature or overnight at 4°C.
Wash in 1mL100% MeOH at Room temperature for 0h 10m 0s. (1/3)
Wash in 1mL100% MeOH at Room temperature for 0h 10m 0s. (2/3)
Wash in 1mL100% MeOH at Room temperature for 0h 10m 0s. (3/3)
Store at -20°Clong-term (can be months or longer)
Day 1
Wear gloves and treat surfaces for RNAses.
Rehydrate the embryos
Wash embryos in 0.5mL 75% Methanol/25% PBTween, rocking, for 0h 5m 0s at Room temperature in 1.5 mL centrifuge tubes.
Wash embryos in 0.5mL 50% MeOH / 50% PBTween, rocking, for 0h 5m 0s at Room temperature
Wash embryos in 0.5mL 25% MeOH / 75% PBTween, rocking, for 0h 5m 0s at Room temperature
Wash embryos in 0.5mL PBTween, rocking, for 0h 5m 0s at Room temperature (1/3)
Wash embryos in 0.5mL PBTween, rocking, for 0h 5m 0s at Room temperature (2/3)
Wash embryos in 0.5mL PBTween, rocking, for 0h 5m 0s at Room temperature (3/3)
Optional: Bleach embryos in 0.5mL freshly-made 3% H2O2 + 1.79millimolar (mM)KOH for up to 0h 5m 0s. Leave the tube caps open and monitor bleaching.
Rinse in0.5mL PBTween (1/2)
Rinse in0.5mL PBTween (2/2)
Permeabilize tissue. Option 1: proteinase K - proceed to step 6.1. Option 2: acetone - proceed directly to step 6.3.
Option 1: Digest with 1mL 10µg Proteinase K in PBTween at Room temperature for 0h 5m 0s or 0h 20m 0s or 0h 30m 0s
Refix tissue in 0.5mL 4% PFA, rocking, at Room temperature for 0h 20m 0s
Option 2: Incubate in 1mL 80% acetone/ 20% diH2O at Room temperature for 0h 20m 0s.
Wash in 0.5mL PBTween, rocking, at Room temperature for 0h 5m 0s (1/3)
Wash in 0.5mL PBTween, rocking, at Room temperature for 0h 5m 0s (2/3)
Wash in 0.5mL PBTween, rocking, at Room temperature for 0h 5m 0s (3/3)
Incubate in 250µL prehybe in hybe oven set to 65°C, rocking, for at least 4h 0m 0s
Incubate with (0.1-1μg/mL) probe diluted in 250µL warmed prehybe , 65°C, rocking.
Day 2
Remove probes. Probes can be stored at -20°C and reused up to 3 times.
Post-hybridization washes
Wash in 0.5mL 100 % (50% 5x SSC / 50% formamide) for 0h 10m 0s at 75°C rocking
Wash in 0.5mL 25% 0.2x SSC / 75% PBTween for 0h 10m 0s at 65Room temperature rocking
Wash in 0.5mL 100% PBTween for 0h 10m 0s at 65Room temperature rocking
Wash in 0.5mL 75% (50% 5x SSC / 50% formamide) / 25% 2x SSC for 0h 10m 0s at 75°C rocking
Wash in 0.5mL 50% (50% 5x SSC / 50% formamide) / 50% 2x SSC for 0h 10m 0s at 75°C rocking
Wash in 0.5mL 25% (50% 5x SSC / 50% formamide) / 75% 2x SSC for 0h 10m 0s at 75°C rocking
Wash in 0.5mL 2x SSC for 0h 10m 0s at 75°C rocking
Wash in 0.5mL 0.2x SSC for 0h 15m 0s at 75°C rocking
Wash in 0.5mL 0.2x SSC for 0h 15m 0s at 75°C rocking
Wash in 0.5mL 75% 0.2x SSC / 25% PBTween for 0h 10m 0s at 65Room temperature rocking
Wash in 0.5mL 50% 0.2x SSC / 50% PBTween for 0h 10m 0s at 65Room temperature rocking
Incubate in 0.5mLblock for at least 2h 0m 0s Room temperature , rocking
Incubate 4h 0m 0s``4°Cwith 0.5mL 1:5000 sheep AP-conjugated anti-DIG Fab fragments (or 1:2000 sheep AP-conjugated anti-FLU Fab fragments)
Day 3
Remove antibody. Antibody can be stored at 4°C and reused up to 3 times.
Post-antibody washes
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (1/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (10/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (2/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (3/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (4/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (5/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (6/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (7/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (8/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (9/10)
Make fresh NTMT buffer. Mix 1mL 1Molarity (M)Tris 9.5, 200µL``5Molarity (M)NaCl, 500µL``1Molarity (M) MgCl2, 50µLTween20 and 8.25mLwater
Equilibrate embryos in 0.5mLNTMT buffer for 0h 5m 0sat Room temperature(1/2)
Equilibrate embryos in 0.5mLNTMT buffer for 0h 5m 0sat Room temperature(2/2)
Transfer embryos to multiwell culture plate (keep the tubes)
Wash in 1mL NTMT buffer for 0h 5m 0sat Room temperature
Prepare fresh stain solution. Choose one of the following:
NBT/BCIP - Indigo stain: Add 4.5µL NBT and 3.5µL BCIP to NTMT buffer. Protect from light. Jump to step 16.
FastRed - Red stain - Dissolve buffer tablet(s) in 1mL/tablet dH2O and sonicate 0h 5m 0s. Dissolve FastRed tablet(s) in buffer and sonicate for 0h 5m 0s. Jump to step 16.
DAB - brown stain - Diaminebenzidine requires a peroxidase-conjugated antibody. Prepare 1x
FR/BCIP - cyan stain - Dissolve buffer tablet(s) in 1mL/tablet dH2O and sonicate 0h 5m 0s. Dissolve FastRed tablet(s) in buffer and sonicate for 0h 5m 0s. Add 3.5µL BCIP and 5.6µL FastRed to fresh NTMT. Vortex and let sit upright for 0h 5m 0s
Replace NTMT in culture plates with 1.5mL 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
Transfer embryos back to tubes. Protect from light in this and all subsequent steps.
Incubate 0h 30m 0s, rocking, in 0.1Molarity (M) glycine HCl 2.2 plus 0.1% Tween at Room temperature to remove first antibody.
Wash away glycine
Wash in 0.5mL PBTween for 0h 5m 0s at Room temperature, rocking (1/4)
Wash in 0.5mL PBTween for 0h 5m 0sat Room temperature, rocking (2/4)
Wash in 0.5mL PBTween for 0h 5m 0sat Room temperature, rocking (3/4)
Wash in 0.5mL PBTween for 0h 5m 0sat Room temperature, rocking 4/4)
Incubate embryos in 100µL preabsorbed sheep AP-conjugated anti-DIG Fab fragments at a 1:2000 dilution in block. You can reuse the antibody 3x. Rock for 2h 0m 0s Room temperatureor 2h 0m 0s``4°C.
Day 5
Remove antibody. Antibody can be stored at 4°C and reused up to 3 times.
Post-antibody washes
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (1/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (10/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (2/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (3/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (4/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (5/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (6/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (7/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (8/10)
Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (9/10)
Make fresh NTMT buffer. Mix 1mL 1Molarity (M)Tris 9.5, 200µL``5Molarity (M)NaCl, 500µL``1Molarity (M) MgCl2, 50µLTween20 and 8.25mLwater
Equilibrate embryos in 0.5mLNTMT buffer for 0h 5m 0sat Room temperature(1/2)
Equilibrate embryos in 0.5mLNTMT buffer for 0h 5m 0sat Room temperature(2/2)
Transfer embryos to multiwell culture plate (keep the tubes)
Wash in 1mL NTMT buffer for 0h 5m 0sat Room temperature
Prepare fresh stain solution. Choose one of the following:
NBT/BCIP - Indigo stain: Add 4.5µL NBT and 3.5µL BCIP to NTMT buffer. Protect from light. Jump to step 27.
FastRed - Red stain - Dissolve buffer tablet(s) in 1mL/tablet dH2O and sonicate 0h 5m 0s. Dissolve FastRed tablet(s) in buffer and sonicate for 0h 5m 0s. Jump to step 27.
VectorRed - Red/yellow stain - To 5mL of 0.1Molarity (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.
DAB - brown stain - Diaminebenzidine requires a peroxidase-conjugated antibody. Prepare 1x
FR/BCIP - cyan stain - Dissolve buffer tablet(s) in 1mL/tablet dH2O and sonicate 0h 5m 0s. Dissolve FastRed tablet(s) in buffer and sonicate for 0h 5m 0s. Add 3.5µL BCIP and 5.6µL FastRed to fresh NTMT. Vortex and let sit upright for 0h 5m 0s. Jump to step 27.
Replace NTMT in culture plates with 1.5mL 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 0.5mL 4% PFA, rocking, at Room temperaturefor 0h 20m 0s
Wash in 0.5mLPBTween, rocking, at Room temperaturefor 0h 5m 0s(1/3)
Wash in 0.5mLPBTween, rocking, at Room temperaturefor 0h 5m 0s(2/3)
Wash in 0.5mLPBTween, rocking, at Room temperaturefor 0h 5m 0s(3/3)
Prepare embryos for glycerol imaging
Wash embryos in 1mL 30% glycerol / 70% PBTween at Room temperature for 0h 10m 0s while rocking and covered in foil.
Wash embryos in 1mL 50% glycerol / 50% PBTween at Room temperature for 0h 10m 0s while rocking and covered in foil.
Wash embryos in 1mL 80% glycerol / 20% PBTween at Room temperature for 0h 10m 0s while rocking and covered in foil.
