I have a dual reporter mouse that expresses RFP and GFP under two promoters. I would like to also do IHC on this tissue but the antigen retrieval step (heat) denatures the GFP. Moreover, if I skip the fixing and antigen retrieval step then the tissue does not preserve. I am using cryosectioning to section the tissue. Has anyone experienced a similar issue? Any suggestions would be much appreciated.
Hello, Gregory! What fixing agent you used for your material?
Did you tried to make a cryosection from fresh tissue?
And, what is less probably: try to make a homogenate from organs of interest and check their fluorescence. There is a small chance, that GFP expression simply not going well.
Like Dmitry, I'm wondering what fixative you're using, not all fixations require an antigen retrieval step, so you might be able to go from the fixation immediately to the antibody incubation. Did the fluorescent markers express correctly?
We use GFP tacked Virus. After 4% PFA perfusion the GFP-immuno works well. We use Invetrogen's rabbit-Anti-GFP-Alexa488 (ThermoScientific A21311) . You can use this antibody for immunfluorescence as well as for DAB-Immunno without antigene retrieval.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue