[Histonet] FISH experts, I need your help!

From:Kim Merriam

Hello,I am currently working on a FISH project (not the swimming kind) and I need some help.I have the FISH procedure working beautifully on cytospin preps of cultured cells and am now trying to get the same probe to work in FFPE archival human tumors.I am using a DAKO probe (dual color, DNA probe, split signal) and the DAKO Histology FISH kit.  Here are the conditions that work on the cytospins (after fixation):	1. denature for 5 minutes @ 84C	2. hybridize overnight @ 43C	3. stringency wash @ 63CNow, I am going with the assumption that the above conditions will not change for FFPE tissue (although I am not sure that this is true) and I am trying to work out the pretreatment conditions (to expose the nuclei).  I am using the reagents that are in the DAKO Histology FISH kit and I am pretreating as follows (as stated in the DAKO kit protocol):	1. heat in 99C waterbath for 10 mintues with DAKO pretreatment buffer (MES)=0A	2. digest in DAKO pepsin for 5-60 minutes (I have tried several different incubation times) @ 37CWhat I am seeing is a bunch of "dots", both red and green and they all appear to be colocalized (not all of them should be colocalized), and these "dots" don't all appear to be specifically in the nuclei, so I am guessing it is either autofluorescence or non-specific staining, but I am not sure.I am wondering if I should try some different types of preconditions (I have read many journal articles on FISH and there are a million different ways that people pretreat their FFPE tissues) or should I start playing around with my denature, hybridization and/or stringency conditions.Any words of wisdom out there?Kim Kim Merriam, MA, HT(ASCP)Cambridge, MA      
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