TITLE: Whole Mount In Situ Hybridization in Zebrafish
AUTHORS: D R Hammond-Weinberger

[DESCRIPTION]
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.

[STEPS]
SECTION: Day 1
3. Wear gloves and treat surfaces for RNAses.
 

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4.1. Wash embryos in 0.5 mL 75% Methanol/25% PBTween, rocking, for 5 min at Room temperature  in 1.5 mL centrifuge tubes.
 

 
 

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4.2. Wash embryos in 0.5 mL 50% MeOH / 50% PBTween, rocking,  for 5 min at Room temperature 

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4.3. Wash embryos in 0.5 mL 25% MeOH / 75% PBTween, rocking,  for 5 min  at Room temperature 

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4. Rehydrate the embryos
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4.4. Wash embryos in 0.5 mL PBTween, rocking,  for 5 min  at Room temperature  (1/3)


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4.5. Wash embryos in 0.5 mL PBTween, rocking,  for 5 min  at Room temperature  (2/3)

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4.6. Wash embryos in 0.5 mL PBTween, rocking,  for 5 min  at Room temperature (3/3)

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6. Permeabilize tissue. Option 1: proteinase K -  proceed to step 6.1.  Option 2: acetone - proceed directly to step 6.3.
 


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6.1. Option 1: Digest with 1 mL 10 µg  Proteinase K in PBTween at Room temperature  for 5 min or 20 min  or 30 min . Proceed to step 6.2

 

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6.2. Refix tissue in 0.5 mL 4% PFA, rocking, at Room temperature  for 20 min . Skip to step 6.4.

 

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6.4. Wash in 0.5 mL  PBTween, rocking, at Room temperature  for 5 min  (1/3)

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6.5. Wash in 0.5 mL  PBTween, rocking, at Room temperature  for 5 min  (2/3)
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6.6. Wash in 0.5 mL  PBTween, rocking, at Room temperature  for 5 min (3/3)
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7. Incubate in 250 µL prehybe in hybe oven set to 65 °C, rocking, for at least 240 min 

 

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8. Incubate with (0.1-1μg/mL) probe diluted in 250 µL warmed prehybe  , 65 °C, rocking.
 


SECTION: Day 2
10. Post-hybridization washes
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9. Remove probes. Probes can be stored at -20 °C  and reused up to 3 times.

SECTION: Day 2
10.1. Wash in 0.5 mL 100 % (50% 5x SSC / 50% formamide) for 10 min at 75 °C rocking

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10.2. Wash in 0.5 mL 75%  (50% 5x SSC / 50% formamide) / 25% 2x SSC for 10 min at 75 °C rocking
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10.3. Wash in 0.5 mL 50% (50% 5x SSC / 50% formamide) / 50% 2x SSC for 10 min at 75 °C rocking
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10.4. Wash in 0.5 mL 25% (50% 5x SSC / 50% formamide) / 75% 2x SSC for 10 min at 75 °C rocking
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10.5. Wash in 0.5 mL 2x SSC for 10 min at 75 °C rocking
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10.6. Wash in 0.5 mL 0.2x SSC for 15 min at 75 °C rocking
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10.7. Wash in 0.5 mL 0.2x SSC for 15 min at 75 °C rocking
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10.8. Wash in 0.5 mL 75% 0.2x SSC / 25% PBTween for 10 min at 65 Room temperature rocking
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10.9. Wash in 0.5 mL 50% 0.2x SSC / 50% PBTween for 10 min at 65 Room temperature rocking
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10.10. Wash in 0.5 mL 25% 0.2x SSC / 75% PBTween for 10 min at 65 Room temperature rocking
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10.11. Wash in 0.5 mL 100% PBTween for 10 min at 65 Room temperature rocking
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11. Incubate in 0.5 mLblock for at least 120 min Room temperature , rocking
 


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12. Incubate 240 min4 °Cwith 0.5 mL 1:5000 sheep AP-conjugated anti-DIG Fab fragments (or 1:2000 sheep AP-conjugated anti-FLU Fab fragments)

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13. Remove antibody. Antibody can be stored at 4 °C and reused up to 3 times.

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14. Post-antibody washes
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14.1. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (1/10)

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14.2. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (2/10)
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14.3. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (3/10)
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14.4. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (4/10)
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14.5. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (5/10)
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14.6. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (6/10)
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14.7. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (7/10)
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14.8. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (8/10)
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14.9. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (9/10)
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14.10. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (10/10)
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15. Make fresh NTMT buffer. Mix 1 mL 1 Molarity (M)Tris pH 9.5, 200 µL5 Molarity (M)NaCl, 500 µL1 Molarity (M) MgCl2, 50 µLTween20 and 8.25 mLwater

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15.1. Equilibrate embryos in 0.5 mLNTMT buffer for 5 minat Room temperature(1/2)


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15.2. Equilibrate embryos in 0.5 mLNTMT buffer for 5 minat Room temperature(2/2)
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16. Transfer embryos to multiwell culture plate (keep the tubes)
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17. Wash in 1 mL NTMT buffer for 5 minat Room temperature

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18. Prepare fresh stain solution.
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18.1. Add 4.5 µL NBT and 3.5 µL BCIP to NTMT buffer. Protect from light.

 

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18.2. Replace NTMT in culture plates with 1 mL of the freshly prepared NBT/BCIP stain solution.

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18.3. Cover with foil
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18.4. 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.
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18.5. Stop the reaction by rinsing in 2 mL PBTween 

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19. Fix tissue after staining
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19.1. Transfer embryos back to their tubes
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19.2. Fix embryos in 0.5 mL 4% PFA, rocking, at Room temperaturefor 20 min

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19.3. Wash in 0.5 mLPBTween, rocking, at Room temperaturefor 5 min(1/3)

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19.4. Wash in 0.5 mLPBTween, rocking, at Room temperaturefor 5 min(2/3)
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19.5. Wash in 0.5 mLPBTween, rocking, at Room temperaturefor 5 min(3/3)
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6.3. Option 2: Incubate in 1 mL 80% acetone/ 20% diH2O at Room temperature for 20 min.

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6.7. Separate embryos into designated tubes (example: sense vs. anti-sense tubes) if not done previously.
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20. Prepare embryos for glycerol imaging
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20.1. Wash embryos in 1 mL 30% glycerol / 70% PBTween at Room temperature for 10 min while rocking and covered in foil.

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20.2. Wash embryos in  1 mL 50% glycerol / 50% PBTween at Room temperature for 10 min while rocking and covered in foil.
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20.3. Wash embryos in  1 mL 80% glycerol / 20% PBTween at Room temperature for 10 min while rocking and covered in foil.
SECTION: Tissue Prep
1. Dechorionate embryos, if needed.
SECTION: Tissue Prep
2. Fix embryos in 500 µL4% paraformaldehyde for 120 min at Room temperature or overnight at 4 °C.

SECTION: Tissue Prep
2.1. Wash in 1 mL100% MeOH at Room temperature for 10 min. (1/3)

SECTION: Tissue Prep
2.2. Wash in 1 mL100% MeOH at Room temperature for 10 min. (2/3)
SECTION: Tissue Prep
2.3. Wash in 1 mL100% MeOH at Room temperature for 10 min. (3/3)
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2.4. Store at -20 °Clong-term (can be months or longer)

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5. Optional: Bleach embryos in0.5 mLfreshly-made 3% H2O2 + 1.79 millimolar (mM)KOH for up to 5 min. Leave the tube caps open and monitor bleaching. 

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5.1. Rinse in 0.5 mL PBTween (1/2)

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5.2. Rinse in 0.5 mL PBTween (2/2)