Methods and Techniques Discussion => Immunohistochemistry (IHC) => Topic started by: SCarolina on August 31, 2015, 11:27:32 AM

Title: Epitope blocked by endogenous Antibody
Post by: SCarolina on August 31, 2015, 11:27:32 AM
I am using IHC to detect viral antigen in frozen, acetone-fixed sections.

I have problems with low sensitivity of detection in tissues from animals that have already developed endogenous antibodies against the viral proteins i am trying to visualize (i.e. late phase infection).

i.e. i am searching for an "epitope retrieval" system that could potentially dissolve binding of endogenous antibody present in the tissue to facilitate subsequent detection of antigen by IHC -preferably something that would work with the acetone fixation I am currently using (although, any suggestions are much appreciated!)

Carolina

Title: Re: Epitope blocked by endogenous Antibody
Post by: gula on August 31, 2015, 01:49:04 PM
I would rather try to increase the sensitivity of your detection system. Increase antibody-concentration, prolong incubation, ad an addtional step in the procedure; Also HIER can improve the sensitivity although the tissue isn't formaldehyde fixed.

Have you done an IHC to prove the theory about bound endogenous antibodies? eg with Fitc-conjugated Anti-IgG.

just some thoughts.
gula
Title: Re: Epitope blocked by endogenous Antibody
Post by: VTimberlake on October 25, 2015, 11:37:01 PM
It is the low affinity and sensitivity that block your epitope. It is advised to change into a new antibody which can meet the needs of experiment such as FMR1NB Antibody.