(Source: Holley Lab from Zebrafish Book 5th Edition)
Fixation
1. Fix embryos with 4% PFA/PBS overnight at 4°C.
2. Wash 2 x PBS, 5' each at room temperature (RT).
3. Dechorionate embryos (easiest at this point).
4. Transfer embryos to 25%, 50% and 75% MeOH for 5 minutes each.
5. Then 100% methanol (MeOH), replace with fresh methanol after 5 minutes.
6. Put at -20°C overnight (or one hour).
7. 5 minutes 75% MEOH in PBST at room temperature.
8. 5 minutes 50% MeOH in PBST at room temperature.
9. 5 minutes 25% MeOH in PBST at room temperature.
10. 2 x PBST, 5 minutes each at room temperature.
11. Fix 20 minutes 4% PFA/PBS at room temperature.
12. 2 x PBST, 5 minutes each at room temperature.
Proteinase and Postfixation
Digest with proteinase K (5 µg/ml in PBST) at room temperature for 5 to 12 minutes depending on the developmental stage (younger stages are more sensitive). Time also depends also upon the batch of enzyme, which should be tested with each new batch.
Rinse briefly in PBST, wash 1 x 5 minutes in PBST.
Fix as above (4% PFA/PBS, 20 min).
Wash as above (2 x PBST).
Prehybridization
Incubate 5 minutes at 65°C in HYB-. Do the HYB- and HYB+ at 55°C if you are also doing the antibody protocol.
Prehybridize at 65°C for 1 hour up to 2 days in HYB+.
Hybridization
Remove all but 50 µl of the preHYB+ (without letting the embryos touch air).
Add 2 µl of each probe (DIG- and Flourescein - labeled probes) to the samples.
Cover tubes in aluminum foil.
Incubate overnight at 65°C (or 55°C).
Probe Removal
Note that the washes from here on lack detergent. This appears to help the staining reactions but does cause the embryos to become rather sticky.
Remove probe.
2 x 30 minutes 65oC (or 55°C) 50% Formamide/2xSSCT.
1 x 15 minutes 65oC (or 55°C) 2xSSCT.
1 x 30 minutes 65oC (or 55°C) 0.2xSSC.
Anti-Fl Antibody Incubation
Block for at least 1 hour at room temperature with 150 mM maleic acid, 100mM NaC1 (pH 7.5) plus blocking reagent (2% Boehringer Blocking Reagent).
Add anti-FL POD as supplied by Boehringer at a 1:500 dilution in above solution.
Incubate 4 hours at room temperature or 4°C overnight.
Wash 4 x 20 minutes in 1x maleic acid buffer and 2 x 5 minutes with PBS.
Detection Of Fluorescein-Labeled Probe
Incubate 45 to 60 minutes in TSA Plus Fluorescein Solution. (Spin down TSA substrate before making the staining solution. For the reaction, dilute tyramide reagent 1:50 in amplification diluent buffer.)
10 min. each in 30%, 50%, 75% and 100% methanol in PBS.
Change to 1% H202 in 100% methanol and incubate 30 min.
10 minutes each in 75%, 50% and 30% methanol in PBS. Then 2 x 10 minutes in PBS. It is important that all methanol is removed.
Anti-Dig Antibody Incubation
Block for at least 1 hour at room temperature with 150 mM maleic acid, 100 mM NaC1 (pH 7.5) plus blocking reagent (2% Roche Blocking Reagent).
Add anti-DIG POD as supplied by Boehringer at a 1:1000 dilution in above solution.
Incubate 4 hours at room temperature or 4°C overnight.
Wash 4 x 20 hours in 1x maleic acid buffer and 2 x 5 minutes with PBS.
Detection Of Dig-Labeled Probe
Incubate 45 to 60 minutes in TSA Plus Cy3 Solution. (Spin down TSA substrate before making the staining solution. For the reaction, dilute tyramide reagent 1:50 in amplification diluent buffer.)
Wash 3 x 10 minutes in PBST.
Propidium Iodide Staining
Wash 2 x 5 minutes in 2 x SSC.
Incubate embryos in 50 µl 2xSSCT with 10 µl RNAse (for a 100 µg/ml final concentration) for 30 minutes at 37°C.
Wash 6 x 3 minutes 2xSSC at room temperature.
Add 0.33 µl 1 mg/ml PPI to 1000 µl 2xSSC and stain for 8 min.
Wash 6 x 3 minutes 2xSSC at room temperature.
Fix 20 minutes 4% PFA/PBS at room temperature.
Wash 2 x 5 minutes PBST at room temperature.
Mounting
10 minutes each in 25% and 50% glycerol in PBST. Clear over night in 75% glycerol.
Carefully dissect and mount the sample. Yolk will cause significant background fluorescence.
Solutions:
PBST: PBS plus 0.1% Tween
SSCT: SSC plus 0.1% Tween
HYB-: 50% formamide, 5xSSC, 0.1% Tween-20
HYB+ HYB-:
5 mg/ml torula (yeast) RNA
50 μg/ml heparin
The torula RNA is prepared by proteinase K digestion of RNA with subsequent phenol-, phenol-chloroform-, and chloroform-extractions. The RNA is precipitated and dissolved in DEPC-treated water.
Tyramide Reagent Solution:
Use a new tube of lyophilized Tyramide reagent, add 60 μl of DMSO, aliquot into 10 μl aliquots and store at -20ºC, in desiccant, protected from light.
Note Regarding PFA:
Make 4% PFA and immediately make 2 ml aliquots and freeze at -20ºC. For the initial fixation, use PFA that has never been previously thawed. For subsequent fixations, you can use PFA that has been previously thawed. The initial fixation appears to be critical for successful staining with this protocol.
This protocol was originally published by:
Jülich D, Lim C-H, Round J, Nicolaije C, Davies A, Schroeder J, Geisler R, Consortium TS, Lewis J, Jiang Y-J, Holley SA. (2005). beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation. Dev Biol 286:391-404.