Well I'm pretty stumped, I just started doing in situ which is not a common technique in our lab. The first couple of times everything went great, the third time the staining was specific but just lighter. This time there was absolutely no staining. I haven't changed the technique, tissue, probe, etc. Nearly everything I have is aliquoted in the freezer and I used all new aliquots this week. The only solutions that aren't aliquoted are the ethanols, PBS/DEPC, DEPC water, and the ingredients for the NTMT. Is this most likely RNAse contamination?? What would you do to troubleshoot. I guess I will start with dumping all my solutions that aren't aliquoted? SOOOOO FRUSTERATED!!