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

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

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4. Rehydrate the embryos
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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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4.4. 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 

 

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

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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.
 

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9. Remove probes. Probes can be stored at -20 °C  and reused up to 3 times.

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10. Post-hybridization washes
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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. Choose one of the following:
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18.1. NBT/BCIP - Indigo stain: Add 4.5 µL NBT and 3.5 µL BCIP to NTMT buffer. Protect from light. Jump to step 16.

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18.2. FastRed - Red stain - Dissolve buffer tablet(s) in 1 mL/tablet dH2O and sonicate 5 min. Dissolve FastRed tablet(s) in buffer and sonicate for 5 min. Jump to step 16.
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18.3. DAB - brown stain - Diaminebenzidine requires a peroxidase-conjugated antibody. Prepare 1x   in DAB buffer. 
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18.4. FR/BCIP - cyan stain - Dissolve buffer tablet(s) in 1 mL/tablet dH2O and sonicate 5 min. Dissolve FastRed tablet(s) in buffer and sonicate for 5 min. Add 3.5 µL BCIP  and 5.6 µL FastRed to fresh NTMT. Vortex and let sit upright for 5 min
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19. Replace NTMT in culture plates with 1.5 mL of the freshly prepared  stain solution.
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19.1. Cover with foil
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19.2. 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.
SECTION: Day 4
21. Incubate 30 min, rocking, in 0.1 Molarity (M) glycine HCl pH 2.2 plus 0.1% Tween at Room temperature to remove first antibody.

SECTION: Day 4
22. Wash away glycine
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23. 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 120 min Room temperatureor 120 min4 °C.
 






SECTION: Day 4
22.1. Wash in 0.5 mL PBTween for 5 min at Room temperature, rocking (1/4)

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22.2. Wash in 0.5 mL PBTween for 5 minat Room temperature, rocking (2/4)

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22.3. Wash in 0.5 mL PBTween for 5 minat Room temperature, rocking (3/4)

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22.4. Wash in 0.5 mL PBTween for 5 minat Room temperature, rocking 4/4)

 

SECTION: Day 5
26. 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
SECTION: Day 5
26.1. Equilibrate embryos in 0.5 mLNTMT buffer for 5 minat Room temperature(1/2)
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26.2. Equilibrate embryos in 0.5 mLNTMT buffer for 5 minat Room temperature(2/2)
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27. Transfer embryos to multiwell culture plate (keep the tubes)
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28. Wash in 1 mL NTMT buffer for 5 minat Room temperature
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29. Prepare fresh stain solution. Choose one of the following:
 

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29.1. NBT/BCIP - Indigo stain: Add 4.5 µL NBT and 3.5 µL BCIP to NTMT buffer. Protect from light. Jump to step 27.

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29.2. FastRed - Red stain - Dissolve buffer tablet(s) in 1 mL/tablet dH2O and sonicate 5 min. Dissolve FastRed tablet(s) in buffer and sonicate for 5 min. Jump to step 27.

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29.3. VectorRed - Red/yellow stain - To 5 mL of 0.1 Molarity (M) Tris-HCl  pH 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.

 

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29.4. DAB - brown stain - Diaminebenzidine requires a peroxidase-conjugated antibody. Prepare 1x   in DAB buffer. Jump to step 27.

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29.5. FR/BCIP - cyan stain -  Dissolve buffer tablet(s) in 1 mL/tablet dH2O and sonicate 5 min. Dissolve FastRed tablet(s) in buffer and sonicate for 5 min. Add 3.5 µL BCIP  and 5.6 µL FastRed to fresh NTMT. Vortex and let sit upright for 5 min. Jump to step 27.
 


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30. Replace NTMT in culture plates with 1.5 mL of the freshly prepared stain solution.
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30.1. Cover with foil
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30.2. 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.
SECTION: Day 4
20. Transfer embryos back to tubes. Protect from light in this and all subsequent steps.
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31. Fix tissue after staining
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31.1. Transfer embryos back to their tubes
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31.2. Fix embryos in 0.5 mL 4% PFA, rocking, at Room temperaturefor 20 min

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31.3. Wash in 0.5 mLPBTween, rocking, at Room temperaturefor 5 min(1/3)
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31.4. Wash in 0.5 mLPBTween, rocking, at Room temperaturefor 5 min(2/3)
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31.5. Wash in 0.5 mLPBTween, rocking, at Room temperaturefor 5 min(3/3)
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24. Remove antibody. Antibody can be stored at 4 °C and reused up to 3 times.
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25. Post-antibody washes
SECTION: Day 5
25.1. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (1/10)
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25.2. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (2/10)
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25.3. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (3/10)
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25.4. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (4/10)
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25.5. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (5/10)
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25.6. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (6/10)
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25.7. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (7/10)
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25.8. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (8/10)
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25.9. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (9/10)
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25.10. Wash in 0.5 mL PBTween for 10 minat Room temperature, rocking (10/10)
 

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32. Prepare embryos for glycerol imaging
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32.1. 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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32.2. 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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32.3. 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)
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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 in 0.5 mL freshly-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 in0.5 mL PBTween (1/2)

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5.2. Rinse in0.5 mL PBTween (2/2)
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6.3. Option 2: Incubate in 1 mL 80% acetone/ 20% diH2O at Room temperature for 20 min.