IHC with monoclonal Ab on mouse tissue

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heathermj
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IHC with monoclonal Ab on mouse tissue

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?

Sandy
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heathermj wrote:I am using a

heathermj wrote:

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?

Could you provide more information regarding the assay and reagents you are using? Is this and IHC experiment or FACS?

heathermj
heathermj's picture
Sandy wrote:heathermj wrote:

Sandy wrote:

heathermj wrote:
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?

Could you provide more information regarding the assay and reagents you are using? Is this and IHC experiment or FACS?

This is IHC on paraffin embedded tissue.

Roshan
Roshan's picture
heathermj wrote:I am using a

heathermj wrote:

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 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.

Paul Weinreb
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Heather,

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.

David
David's picture
heathermj wrote:I am using a

heathermj wrote:

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?

For IHC Dako ARC kit will overcome this problem - have this and it works well.