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Permeabilize embryos, hybridize probes, destroy native peroxidase activity,bind anti-Fluorescein-peroxidase, develop Tyr-Fluorescein, destroy peroxidase activity, bind anti-DIG-peroxidase, develop Tyr-Cy3, destroy peroxidase activity, bind anti-DNP-peroxidase, develop Tyr-Cy5, H2O2 treat to reduce background.
Embryo preparation:
1. Fix embryos overnight at 4˚c in 4% PFA/1X PBS
2. Wash twice in PBST
3. Dechorionate embryos using a pair of watchmakers forceps
4. Dehydrate with a series of methanol/PBST solutions (25%, 50%, 75% methanol mixed with PBST), then twice with 100% methanol. Shake 3-5 minutes in each solution.
5. Store the embryos in 100% methanol at -20˚C
Day 1:
1. Rehydrate the embryos through a methanol/PBST series (75%, 50%, 25% methanol mixed with PBST) 3-5 minutes per wash.
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11. Remove probes, and save them for reuse. (store @ -80˚C)
12. Wash 5 mins in "5X" “5X” @ 68-70˚C
13. Wash 5 mins in 3:1 5X:2X @ 68-70˚C
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15. Wash 5 mins in 1:3 5X:2X @ 68-70˚C
16. Wash 5 mins in "2X" “2X” @ 68-70˚C
17. Wash 3 X 20 mins in 0.2X SSC, 0.25% tween-20 @ 68-70˚C
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29. Shake one hour in the dark, tubes upright. Don't Don’t exceed one hour. From here on out longer steps are done in the dark (ie- a covered box), though the dyes are quite photo-stable, so I do the shorter washes under normal lighting.
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Fluorescein Labeling Mix: Roche, cat# 11685619910
20XNTP mix: 100mM NTP's NTP’s obtained from Amersham Bioscience (cat# 27202501).
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Test pH, should be 6.0-6.5
store @ 4˚c
"5X"“5X”: (5X SSC, 50% formamide, 0.25% tween-20)
100ml formamide 50ml 20X SSC 2.5ml 20%tween 47ml sterile H2O
store at 4˚C
"2X"“2X”: (2x SSC, 0.25% tween-20)
40ml 20X SSC 5ml 20% tween-20 sterile H2O to 400ml
store at room temp
"0“0.2X"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 I’ve had problems with this protocol, I've 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.
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This protocol combined procedures developed in a number of labs, and contains modifiers from Isabelle Manfroid, Macie Walker, and Yi-Lin Yan's Yan’s working protocols, in addition to our own (Jared Coffin Talbot). See also:
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