...
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.
...
54. Wash 4 times in TNT
55. Store fish in * * TNT @4˚C.
Pertinent Materials:
...
TSA Cyanine 5 system: Perkin Elmer Cat# NEL745001KT
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
Antibody preabsorption (optionalAntibody preabsorption (Optional- if desired.):
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.
...
200µl 50mg/ml Heparin 400µl 250mg/ml yeast tRNA 1.9ml 1M citric acid
Test pH à , should be 6.0-6.5
store at @ 4˚c
"5X": (5X SSC, 50% formamide, 0.25% tween-20)
...
4ml 20X SSC 5ml 20% tween-20 sterile H20 to 400ml
store at room temp
Probe Probe synthesis:
DNP Probes: DIG probes: Fluor Probes:
...
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.
Sources:
This protocol combined procedures developed in a number of labs, and contains modifiers from Isabelle Manfroid, Macie Walker, and Yi-Lin Yan's working protocols, in addition to our own (Jared Coffin Talbot). See also:
MCM Welten et al. 2006. ZebraFISH: Fluorescent in situ hybridization protocol and three dimensional imaging of gene expression patterns. Zebrafish v3 (4) 465-476
I Manfroid et al. 2007. Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development_. Development_ v134 (22) 4011-4021
M Lopez et al. 2006. Expression of the somatolactin beta gene during zebrafish embryonic development. Gene Expression Patterns v6 (2) 156-161
T Jowett, YL Yan. 1996. Double fluorescent in situ hybridization to zebrafish embryos. Trends In Genetics v12 (10) 387-389