I am trying to standardize IHC for cFOS and FGFR1 detection on rat brain sections. I use 40 micron sections and have had good results for cFOS detection. But I have not had any positive results with FGFR1 staining. I was wondering if anybody has performed IHC on floating sections for FGFR1 with good results
https://www.abcam.com/protocols/immunostaining-paraffin-frozen-free-floating-protocol
Hi Rashmi Dubey ,
The fixation methods and time of fixation are important when you want to detect some markers as membrane receptors. I guess that you have been reading about it.... I think You should optimize an antigen retrieval protocol for this target.
https://www.abcam.com/protocols/ihc-antigen-retrieval-protocol
Good luck!!
Hi Angel Thanks for the reply. Have you any suggestions for antigen retrieval for floating sections? I use 40 micron sections.
Hi Rashmi Dubey ,
I use PFA for fixation and 30um brain sections. In floating sections, I use heat-induced epitope retrieval with Sodium citrate buffer (10mM, pH6) or commercial SSC from Sigma. I transfer the sections in SSC. If you could do it in a water bath (bain-Marie) it would be great (probe several times 5-10-20 min).... but also You can use an oven (1-2h at 60-70ºC - put inside the oven a container with water to avoid dry your samples... You know...) for doing it ... Once this step ended, You should refresh and incubate with new SSC at RT 10min (once or twice) and continue with your IHC protocol.
The time and the temperature must be optimize according your sample and the type of antibody. Use positive and negative control for your target....You know...
I hope you find it useful!
Best!
Ángel
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining