Hi Seven

Ive attached my protocol for IHC on frozen sections,  Depending on how much of your protein is there with a 1 step staining procedure you may not see much signal. You can always amplify with a biotin labled secondary and Streptavidin HRP.

good luck Caroline 

Hy Seven,

In my experience there is no single protocol for every antybody out there. Depending whether you are using formalin fixed tissues weather embedded in parafin or in freeying medium for cryosections there is a number of protocol you can follow. Here is my paper with detailed protocols we usualy employ when testing new antibodies for optimal conditions. hope this helps.

Cheers!

Chapter Role of microwave heating in antigen retrieval in cryosectio...

Since HIS6 is an extreme positively charged tag, one has to take into account that any prior exposure of the tissue to hydrophobic agents will make the tag buried inside the folded protein.

Hi again,

Forgot to say that if you are using DAB then you should do a hydrogen peroxide step 3% H2O2 in PBS for 20mins after the rehydration series. This will reduce the endogenous peroxidase activity in your tissue.  PS Always run am isotype control at the same time.

Caroline

Follow any published method!

Hi every body,

Thank you all for your kind & helpful answers. Also, recently I found these protocols in Abcam, rockland-inc & IHC world websites which maybe benefit, thought. I do include your kind advises in protocols.

best regards

Seven

Dear Caroline Elizabeth Dunk,

Thank you again for your excellent protocol for IHC on frozen sections. It was very helpful. I stained frozen sections according that protocol & guess what ...The outcome was just perfect and clear.

best wishes for your work & life

Seven