In Situ Hybridization of ZF embryos 1 of 6
IN SITU HYBRIDIZATION OF ZEBRAFISH EMBRYOS:
Use RNase free 1.5 mL eppendorf tubes for incubation. Use The Pipette Pump F37898 from
Bel-Art Products Pequannock, NJ and a glass Pasteur pipette unmodified for taking off liquid or
a glass pipette with the end broken off and smoothed by flame to allow a wider opening to take
up embryos.
Wash with dH2O before using. Add liquid to side of tube, especially after dechorionation, so
embryos do not break. Also do not expose embryos to air or they will degrade. Good stages to
look at first are 5S, 18S, 24S, 30S,48 hr. Take 5 embryos per stage.
Day One (estimated time ~ 5 hrs)
1. Rehydrate embryos with 1 mL as follows (embryos are fixed O/N at 4˚C in 4% PFA in PBS,
then was washed with PBST 2X5’, equilibrated with 4-5 changes of methanol and stored in
100% methanol at 20˚C in glass specimen vials or eppendorf tubes. Embryos can be stored
years like this):
a. 75% methanol + 25% PBST 5 min
b. 50% methanol + 50% PBST 5 min
c. 25% methanol + 75% PBST 5 min
2. Wash embryos with 100% PBST 5 X 5 min. (At any point you can leave embryos in PBST
for longer).
3. Dechorionate embryos. Use Stemi 2000-CS Zeiss dissecting microscope with FOSTEC
Schott-Fostec, LLC EKE Ace I power supply for light. Use small forceps with sharp points
at end. Put embryos in 100% PBST in petri dish and remove chorion by either of 2 methods:
a. Put forceps on both sides and pull in opposite directions
b. “Guide” embryo out of chorion. Be careful, they are fragile!
4. Treat embryos with proteinase K (1 mL/tube) to put holes in membrane so RNA can get in:
Bill Campbell 1/20/03
In Situ Hybridization of ZF embryos 2 of 6
Stock [c] = 1 mg/mL; stored at - 20˚C
Working [c] = 10 ug/mL
therefore, 10 mL for 1 mL (1:100) in RNase free H2O
time = 5-10 min for pre 1S (before 10.5 hr)
50-15 min for pre 16 hr
15-25 min for post 16 hr
15 min for post 16 hr
20 min for 52 hr
25 min for > 25 hr
5. Quick rinse with PBST 2X (1mL/tube)
6. Refix embryos in 4% PFA in PBS (stored at 4˚C) 20 min at RT.
7. Rinse in PBST 5 X 5 min.
8. Prehybridize for 2 hr at 65˚C in 300 uL hyb buffer.
30 min before end of prehyb, prepare probe preheated in 65˚C for 30 min.
stock for antisense probes is already at 1:10 dilution for hybridization, need 1:200 dilution
Therefore, take 190 uL hyb buffer + 10 uL probe for each tube.
9. Discard the prehyb soln, add 200 uL probe (1:200)/tube. Incubate 65˚C, O/N. Meanwhile,
prepare the follow solns and preheat O/N at 65˚C:
a.
b.
c.
d.
75% hyb + 25% 2X SSC
50% hyb + 50% 2X SSC
25% hyb + 75% 2X SSC
100 % 2X SSC
Bill Campbell 1/20/03
In Situ Hybridization of ZF embryos 3 of 6
Day Two (estimated time ~ 4 hr)
1. Wash 2X 30 min with 0.2X SSC 1 mL/tube at 65˚C. 0.2X SSC does not need to be preheated at 65˚C.
2. Room temperature: (1mL/tube)
a.
b.
c.
d.
75% 0.2X SSC + 25% PBST 5 min
50% 0.2X SSC + 50% PBST 5 min
25% 0.2X SSC + 75% PBST 5 min
100% PBST
5 min
3. Prepare 5% sheep serum in 2 mg/mL BSA in PBST: 500 uL/tube (one for blocking step, one
for 2*Ab)
e.g. 1900 uL 2 mg/mL BSA in PBST + 100 uL 100% sheep serum = 2 mL (This is enough
for 2 tubes).
4. Block 500 uL/tube for 1-3 hours @ 4˚C (typically 2-3 hr). Store unused 5% SS that will be
used for 2*Ab at 4˚C until it is ready to be used.
5. Incubate in 1:2000 dilution of anti-Digoxigenin-Alkaline phosphatase conjugated Fab
fragments antibody O/N at 4˚C. 1:2000 dilution or want 0.375 units/mL
e.g. 8 mL 5% SS + 4 uL anti-Digoxigenin-AP
Bill Campbell 1/20/03
In Situ Hybridization of ZF embryos 4 of 6
Day Three (estimated time ~ 4 hr)
1. Wash at RT with 2 mg/mL BSA in PBST (stored at 4˚C between each step) 500 uL/tube (8 X
15 min)
2. Can now stored at 4˚C in PBST O/N if no time for staining or continue.
3. Prepare NTMT (all stock solns are autoclaved!): e.g. for 50 mL:
1M NaCl
1M Tris, pH 9.5
1M MgCl2
Tween 20
Sterile H2O
5 mL
5 mL
2.5 mL
50 mL
37.5 mL
4. Equilibrate 3 X 5 min with NTMT, 1 mL/tube.
5. Stain with BCIP/NBT in dark environment (cover with foil):
BCIP/NBT pre-mix is in 4˚C. Dilute 1:50 in NTMT e.g. 200 uL/10 mL NTMT.
Remove embryos to 12 wells plate with NTMT, then discard NTMT. Do 1 well at a time. It
is OK if embryos are dry for a few seconds.
Add BCIP/NBT 1 mL/well.
Keep in dark (wrapped in foil) at RT. Stain for ~ 30 min to 2 hr or even longer if
necessary. Check every half hour.
6. Stop staining by washing 3X with PBST (3 short washes) and store at 4˚C wrapped in foil.
Take pictures 1 week after staining.
Bill Campbell 1/20/03
In Situ Hybridization of ZF embryos 5 of 6
To Take Picture
- Wash with 100% methanol 3X 20 min or 1X 30 min according to Nishant/Tao 6.28.02
- Wash with PBST 3X quick wash.
- Keep in PBST at 4˚C, wrapped in foil, and take pictures. To take pictures, put some 3% methyl
cellulose on glass decompression slide and put one embryo at a time with PBST onto
slide
Probe recommendations:
- can try 1:400 dilution of probe if 1:200 gives staining ubiquitously
- usually include some 3”UTR so probe is more specific
Solution:
o 1X PBS = 2 L: 16g NaCl, 0.4g KCl, 2.88g Na2HPO4, 0.48g KH2PO4 - pH 7.4
o PBST (1X PBS, 0.1% Tween 20)= 500 mL PBS + 0.5 mL Tween 20. Autoclave.
o Proteinase K 1mg/mL in PBST at - 20˚C.
o Proteinase K 100 mg Boehringer/Roche 745 723
o Hybridization Buffer:
50% formamide
5X SSC
5 mg/mL Torula (yeast) RNA
50 ug/mL heparin
0.1% Tween 20
Gibco BRL 500g 15515-026
Sigma 100g R-6625
Sigma H-7005
for 50 mL:
25 mL formamide
12.5 mL 20X SSC
250 mg torula (yeast) RNA
5 mL 500 ug/mL heparin
50 uL Tween 20
Bring up to 50 mL with Rnase/Dnase free H2O (~7.5 mL H2O).
o 4% Paraformaldehye for 500 mL:
20g PFA in 400 mL PBS. Heat to ~ 70˚C. Add a few drops of 5N NaOH to pH 7.4 and then
add H2O to 500 mL final volume. Filter. Store at -20˚C.
o PBST + 2 mg/mL BSA. For 50 mL:
50 mL PBST
100 mg BSA (Sigma A-8022 or A-2153)
o Sheep Serum: Cat Sigma S2263
Bill Campbell 1/20/03
In Situ Hybridization of ZF embryos 6 of 6
o 20X SSC
NaCl
175.3g
NaCitr
88.2g
H2O
800 mL
14 N HCl until pH = 7.0, then bring up to 1000 mL
Final [c] = 3 M NaCl, 0.3 M NaCitrate
o Gibco Dnase/Rnase free H20 = Cat# 10977-015
o 2*Ab = Roch Enzo Anti-Digoxigenin-AP Fab fragments Cat# 1 093 274
(150 U, 200 uL; stored at 4˚C)
o BCIP/NBT pre-mix = Roche Cat# 1 681 451 (8 mL)
o Hybridization Oven = Robbins Scientific Model 2000 Micro Hybridization Incubator
Bill Campbell 1/20/03