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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunohistochemistry In Situ Hybridization forum
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TSA FISH
Posted by: ping (IP Hidden, New member, 1)
Date: January 2, 2008 02:35AM
Hi I'm new to this forum. Have been experiencing some difficulties with my metaphase FISH, hope to get some help.
Obtained nice signals / low background with FISH after tyramide signal amplification. But got no signals when I repeated the experiment, instead there were spots everywhere on the slide. Tried to get rid of background by repeating again, with thorough washing steps. Background eliminated but signal also lost. After tweaking parameters such as concentration of pepsin / duration of pepsin incubation / concentration of probe / concentration of primary antibody, now I get whole chromosome signals rather than discrete spots (ie, the entire chromosome appears to be labelled). Probe sizes between 100-500 bp Reagents such as pepsin stock, hybridization buffer all prepared by batch and stored in aliquots. Why am I unable to reproduce the signals? Is there a very sensitive step that I may have overlooked / missed out?
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