Dear Christine 

It's look like your processing the tissue by cryosections... Correct???. If so.  It wont required antigen retrivel step, But you need to do permeabilization step ( yes triton x will help you in this step), there r some other methods as well.

We do in our lab for nuclear antigen, incubate section with o.1% triton x  before blocking step and it works. 

One more, it will help you more than any. Check the antibody website, for eg abcam -there will be always users review , posted the working protocol. Most of time this helped us to get the staining instead of beating around the bush. and other option is check the articles any one used your antibody previously and try their protocol. 

All the best

Hi Karthikeyan,

Thank you for your suggestions.

Yes these are processed by cryosections. I have tried with and without 0.1% Triton-X and Tween-20 (I have it at that concentration in my block, and in the block I put the primaries in overnight).

I'm getting better labelling without Triton, so I think it's too harsh and destroying the membrane and antigen.

The microwave protocol is the one recommended by the manufacturer and published, but adult human biopsies are much more robust than embryonic bird.

Thanks,

Christine

Hi Christian

You mean, u have used both triton x and tween together (Just to clarify) ???. Yeah it s true that tritox s a strong detergent, where as tween 20 is mostly recommended in case triton x staining s bad.

Some case, we used enzymatic methods (Pepsin) which is also make membrane permeable. 

No, sorry I was being unclear. I have tried no detergent or 0.1% Triton or 0.1% Tween.

Enzymatic methods may be the way forward - thank you for your suggestion.

Thanks,

Christine