GFP immunohystochemestry PFA issue
Posted 30 September 2010 - 11:11 PM
I need to performe an immunohistochemistry for eGFP detection in not perfrused brain (just frozen in dry ice or fixed soon after been cut and stored a 4 C) and I wish not to eluite my protein after the first wash
Posted 02 October 2010 - 02:06 PM
Posted 03 October 2010 - 09:50 AM
It was like to see a green signal very specific but with very bad green cell morfology :-(
At that point the sections have been frozen but when I de-frozen and fixed them with PFA I found my green much weacker :-( at the moment I'm planing to do an immuno to signail retrive but I do not have much hope to have the resolution I need for the quantification...pre fix brain with PFA or much warse perfuse, make the tissue very difficult to be cut with the criostat, and I scared to loose my samples.....I guess there is no much to do....
Posted 03 October 2010 - 03:18 PM
For autofluorescence try fixing in DMSO/methanol and then clearing in DMSO/Methanol with 2% H2O2.
Posted 04 October 2010 - 05:41 AM
and see if the cellular resolution get better
about the fixation in DMSO/methanol:
1. I supposte DMSO is to crioprotect and methanol as fizative, you meant I should fix my brain in this way, freeze it and then cut it ???
2. is this tretment suitable for GFP detection? I mean because of
the small size of the protein I thought the best choice would be PFA fixation
(because of the reticulum it forms over the proteins to block
them were they are) but I really don't know why methanol would has fixation property.....by the way do you think this would be strong enought for GFP???
Posted 04 October 2010 - 03:21 PM