I am using a monoclonal Ab to detect 11B hydroxylase in mouse testes and am having problems with the 2nd Ab binding non-specifically to the tissue since the 2nd Ab is anti-mouse. Can anyone suggest how I get round this problem?
I am using a monoclonal Ab to detect 11B hydroxylase in mouse testes and am having problems with the 2nd Ab binding non-specifically to the tissue since the 2nd Ab is anti-mouse. Can anyone suggest how I get round this problem?
heathermj wrote:
Could you provide more information regarding the assay and reagents you are using? Is this and IHC experiment or FACS?
Sandy wrote:
This is IHC on paraffin embedded tissue.
heathermj wrote:
I think you need to block the tissue before adding the second antibody. Just like and ELISA assay. After you add the monoclonal Ab and wash block it with 1% milk powder or BSA in PBS. Incubate and wash and then add your secondary antibody. I hope this will solve the problem.
Heather,
Have you tried biotinylating your antibody and using an avidin-based reagent for detection? Alternatively you could directly label the primary antibody with a fluorescent dye (e.g. ALEXA488 from Molecular Probes) and avoid the secondary altogether.
heathermj wrote:
For IHC Dako ARC kit will overcome this problem - have this and it works well.