[Histonet] Need help with FISH staining protocol on frozen tissue
jinhui <@t> uta.edu
Wed Mar 10 00:44:08 CST 2010
Has anyone done FISH staining on mice tissue frozen sections for Y chromosome? I need help with a successful protocol.
Recently I started to do FISH staining on mice liver frozen sections for chromosome Y, but got no positive results even on male samples yet. The probe I used was IDMF1057 Green label Chr Y probe from ID Labs Inc. (www.idlabs.com). They have a protocol online probably for cultured cells (http://www.idlabs.com/product/datasheets/IDMF1057.pdf?PHPSESSID=b39f16507cf76b69495166c651f0b52c) which I haven't tried yet. I followed the following protocol for frozen sections:
1. Fix sections in 4% PFA at 4C for 10 mins, then wash in PBS.
2. Treat the sections with 0.01% Pepsin, 37C 10min, wash in PBS.
3. Dehydrate the slides using 70% Ethanol (2minsX2), 90% Ethanol (2mins X2), 100% Ethanol (5 mins X1).Then Age the slides at 65C for one hour.
4. Mix the probe and hybridization buffer. Denature the probe by incubate it at 65C for 10 mins, then hold it at 37C for 30 - 60 mins.
5. Denature the sections by incubate them in denaturing solution (formamide:2XSSC, 70:30) at 75C for 2 mins.
6. Quench the slides in 4C 70% Ethanol for 4 mins. Dehydrate the slides as step 3.
7. Apply the probe and hybridize at 37C overnight.
8. Immerse the slides in 1XSSC for 5 mins to remove the cover slips.
9. Wash slides in stringency solution (formamide:2XSSC, 50:50) at 45C, 5 mins X2.
10. Wash slides in 1XSSC at 45C, 5minsX2.
11. Wash slides in detergent wash solution at 45C, 5minX1.
12. Rinse slides in PBS.
13. Air dry for a couple of minutes and counterstain with DAPI.
14. Mount the slides and observe the results or keep in 4C for late check.
Are there any problems with this procedure? Please help me to check, or if you could recommend a working protocol you had used, I would really appreciate.
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