Re: [Histonet] repeating AR heat treatment

From:Rene J Buesa

  HIER (Heat Induced Epitope Retrieval) treatment is to "undo" the formaldehyde crosslinking of proteins. The application of the first Ab was specifically binded to their antigenic sites and did nothing to other antigenic sites.
  HIER eliminated crosslinkage to all antigenic sites. If your new primary targets the same antigen as your FITC probably it will not be able to link unless you eliminate that FITC conjugated Ab; they are competing for the same site.
  I don't think that new HIER session will solve this problem.Perhaps immersing the sections in a low pH solution (pH 5) will break the Ag-Ab reaction you obtained with your FITC conjugated Ab. Probably that would be enough. After the treatment (that will affect the quaternary structure of the Ag site) you should place the slide in pH neutral buffer to try to "return" the structure to its natural configuration.
  If you are targeting another Ag sites, you don't need another HIER treatment, all sites should have become "available" to receive the Abs.
  Hope this will help you!
  René J.

"Bobrowitz, Carol"  wrote:
  I have already stained FFPE rat kidney's using FITC with a 2 hour heat treatment in citrate buffer ph 6.0. (the staining was great)

I am now going to restain the same rat kidney slides with an additional primary using DAB as the chromagen. This primary also requires heat treatment.

Am I correct in my thinking that I should repeat the heat treatment again due to the fact that the epitope (binding) sites have already closed after accepting the application of the 1st primary?

Any help will be appreciated. Thank you in advance.

Carol Ann Bobrowitz
Medical College of Wisconsin
Department of Physiology
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