Hi, everyone. I am going to do IHC to stain CD4, CD8, CD3, MHC II in brain sections, and I was told by my superviser that non-fixed section should be used.
But in papers that I had read, PFA fixed sections were used, only in this paper 'The Inflammatory Reaction Following 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Intoxication in Mouse' they used non-fixed section(free frozen section) for MHC II, but PFA fixed section for CD4 et.al.
Can anyone tell me is it true that non-fixed section should be used for staining for lymphocyte? Why is that? Any other cases that non-fixed section should be used?
Many thanks.
Hey, Srinivasa, thanks.
I use antibodies from serotec. The datasheet did not say anything about fixation, I asked their technique support but did not get answer yet.
I am confuse about what harm would PFA fixation would do to the CD4, CD3... I will do antigen retrieval anyway.
Hey, Ariel. Recently I used CD4 and CD8 antibodies to stained mouse skin through IHC (antibodies were purchased from BD). The data sheets indicated only froozen sections, and even frozen section should be fixed. Most antibodies, from Serotec for IHC also stained only froozen sections (for mouse). There is a site (IHC World - http://www.ihcworld.com/_protocols/histology/frozen_section_technique_1.htm) that could help you in the experiments. Good luck! Felipe.
Hi Ariel, did you have any luck with your IHC? I am doing an IHC with non-fixed frozen sections for CD3, CD4 and CD8 but I am not having positive results. I was wondering if you have a protocol for these IHC in frozen sections that you can share with me. it will be a lot of help.
Thanks!
Hi Ariel,
I'm gonna do immunostaining with CD8 antibody and I was wondering if it works with previous fix with 4%PFA. Would you have any protocol?
Would be very helpful!!
- Badges
- Science method