Polyclonal antibodies in immunocytochemistry not working - (Jul/12/2007 )
Hi,
I have been trying to do immunocytochemistry with cyro-sectioned brain tissue and so far have only been able to get my monoclonal antibodies to work. I need both monoclonal and polyclonal so that I can double-label each section for myelin proteins like MBP and also the protein of interest.
The monoclonal antibodies always work. The polyclonal never. I have tried permeablizing with both acetone and methanol for various amounts of time, and have tried several primary polyclonals as well as two different secondary polyclonals. Any advice? Is there anything different that I can do for mono vs polyclonal, and why?
Thanks!
Do antigen retrieval with the sections and then add the primary polyclonal antibody. It can help.
acetone and methanol will fix your sections (histo = sections , cyto = cells or are you doing something else)
try triton X (0.2% in pbs) for ten minutes after the fix
dom
I have been trying to do immunocytochemistry with cyro-sectioned brain tissue and so far have only been able to get my monoclonal antibodies to work. I need both monoclonal and polyclonal so that I can double-label each section for myelin proteins like MBP and also the protein of interest.
The monoclonal antibodies always work. The polyclonal never. I have tried permeablizing with both acetone and methanol for various amounts of time, and have tried several primary polyclonals as well as two different secondary polyclonals. Any advice? Is there anything different that I can do for mono vs polyclonal, and why?
Thanks!
many antibodies are not to use for immunhistochemistry or immuncytochemistry; often, especially if it is a company-provided Ab, there are information about the immuno techniques where this Ab is to use; if there are no information you may try and do an optimization protocol as suggested by other members
Hi,
Thank you so much!! I looked up antigen retrieval protocols, and found one that "frozen section epitope retrival" that uses SDS (and I guess is not really antigen retrieval) and a few other actual antigen retrieval protocols. I tried the SDS since it was the simplest, and it worked beautifully. I am new to IHC so appreciate your help.
Thanks!