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5. Store the embryos in 100% methanol at --20˚C
Antibody preabsorption (optional):
1. Prepare100 embryos of mixed stages with in situ (Day 1) protocol steps 1-7, ending in pre-hyb on day one of the in situ. These can be stored at --20˚c for quite a while. On in-situ Day 2, Day 3, and Day 4, prepare embryos respectively for µFluor µDig, µDNP preabsorption.
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3. Wash once in TBSTB
4. Add TBSTB to the tubes, 400µl times (number of samples plus 1)
5. Add anti-DIG-peroxidase (1:1000) or anti-DNP peroxidase (1:500) or anti-Fluorescein peroxidase (1:5000) to the tubes.
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7. After use, store the embryos in pre-hyb solution. They can be reused several times. Be sure to label what antibody was used on those embryos, and only reuse them for that antibody. To reuse, remove fish from freezer and enter pre-absorption protocol on step 2.
Recipes:
PBST: (1X PBS, 0.25% tween-20)
100ml 10X PBS 12.5ml 20% tween-20 sterile H2O to 1L
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100ml Tris pH 7.5 30ml 5M NaCl 25ml 20% tween-20 sterile H2O to 1L
store at room temp
TBSTB: (TNT with 0.5% Perkin-Elmer blocking powder)
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Store at -20˚C, and once thawed, never refreeze.
Pre-Hyb: (50% formamide, 5X SSC, 100µg/ml yeast RNA, 50µg/ml Heparin, 0.25% tween-20, Citric acid to pH 6.0 [appx 0.02M citric acid final])
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100ml formamide 50ml 20X SSC 2.5ml 20%tween 47ml sterile H2O
store at 4˚C
"2X": (2x SSC, 0.25% tween-20)
40ml 20X SSC 5ml 20% tween-20 sterile H2O to 400ml
store at room temp
"0.2X": (0.2X SSC, 0.25% tween-20)
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The largest factor that dictates whether the in situ will work well is the quality of probes used. When I've had problems with this protocol, I've generally been able to solve them with new, cleaner, probes. When a probe has worked well once, it has continued to work well when reused several times. In my hands, DNP works as well, if not better, than DIG for probe labeling, though Fluorescein labeled probes have the reputation for giving background. Tyr-Fluor and Tyr-Cy3, and Tyr-Cy5 produce comparable signal to noise ratios and good labeling when developed under similar conditions. Cy5 (far red) wont show up under many fluorescent dissecting microscopes, so we generally use this probe primarily to give context to the other two probes. As per the norm with in situ, it is critical that the pH of pre-hyb is around 6-6.5, and it is also critical that the fish never dry out. The dyes show very little photobleaching, even if left in the light overnight. It is critical that the antibodies are not left at room temperature for too long, as this destroys peroxidase activity. Some of the antibodies are shipped at room temperature- they should work, but must be stored at 4 degrees immediately upon receipt.