Dear researchers. I did IHC on GFP-positive tissue section with heat antigen retrieval step, followed by normal protocol. Finally, I didnt managed to view any fluorescence from GFP as viewed before IHC protocol. Any reason why the GFP fluorescence faded? Is it due to the antigen retrieval step destroyed the proteins? Any suggestions to overcome this challenge?
My aim is to track those implanted cells after 3 months implantation in nerve conduit. Thanks in advance!
Gfp degrades at high temperatures. If you have to use antigen retrival to detect another protein, you could try using an anti-gfp with alexaflour 488 secondary to recover the signal.
That was a good suggestion. Thanks. or I should try another method of antigen retrieval.
Yes, but in general the more processing the more you will decrease the GFP signal. Even PFA fixation decreases GFP fluorescence in comparison to live imaging. I would still suggest boosting the signal with the GFP antibody.
I would second Alexander's suggestion. I've personally never had much luck looking at the inherent fluorescent activity of GFP following similar tissue processing steps.
I did fixed in 4%PFA and do frozen section instead of embed in paraffin. Then I directly view under fluorescence microscope. We can observed the GFP fluorensce brightly. It just faded after the IHC protocol.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publications