[Histonet] Question: MHC Class II Immunofluorscent Staining on CNS tissue.

[Vin Penaflor]

Hi, 

 

I am currently having an issue with trying to stain MHC Class II (IaB/AF6
Clone) on mouse brain tissue for confocal microscopy immunofluorescence. I
have been trying to use BD Pharmingens, Biotinylated Anti mouse I-Ab coupled
with a secondary molecular probes streptavidin-594. So far I have not been
able to get the Iab staining to work. I get completely no staining. I am
curious if anyone has a successful protocol with conditions that would
produce a very bright staining on confocal microscopy with minimal
background for this antibody. Any help would be greatly appreciated. I have
tried different perfusion techniques, freezing techniques, fixations, wash
steps, and dilutions of the antibody and so far no luck. If anyone has
conditions or a protocol that works well with this particular antibody on
brain or spinal cord tissue, I would greatly appreciate any input. I have
tried using the primary and secondary antibody on mouse spleen tissue and it
works PERFECTLY, it's just that somehow in the brain I cannot get it to
work. Also if there are better antibodies/secondary from other companies I
should try that would also be helpful! Thanks in advance!