Methods and Techniques Discussion > Immunocytochemistry (ICC)

cytospin permeabilize and blocking serum

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apple:
Hi,

Most people use Triton-X-100 permeabilization, but some people use cold acetone permeabilization. Why?

Usually, when we use secondary antibody for undirectly staining, we use serum (produced from the same animal of secondary antibody) to block unspecific binding. How about direct stain method. for example use FITC/PE conjugated primary antibody,  or biotin labelled primary ab with avidin labelled peroxidase or avidin conjugated FITC/PE. Do I still need to use normal mouse serum block ?

Thanks

ImmunoNYC:
In theory you don't need the serum but I normally use it anyway. It can also potentially act as an Fc blocker. My theory on my blocking buffer is that if it ain't broke don't fix it. My block gives me backgroundless stains so why alter it even if my primary is directly labeled!

Triton-X-100 permeabilization is normally used after a PFA fixation where the proteins in the plasma membrane have been cross-linked thereby not allowing access to the cytosol of the cell. This is a problem when staining an intracellular antigen so one permeabilizes to gain access.

Acetone is an organic "fix" (ketone) which essentially dries out cells and punches "holes" in the membrane by coagulating proteins without altering them chemically. People use it cold IMO  only because it used to be used for RNA extractions and the cold temperature prevents RNase activity. I mostly use it at RT without issues and always have.

apple:
Hi MaximinaNYC,

Many thanks.

I though I should still block the slide. But I don't know which blocking serum I should use? Should I use normal mouse serum or BSA as blocking agent?

Thanks

ImmunoNYC:
What is the species of your primary and what sort of tissue (what species) are you working with?? That should make your decision for you.  

If in doubt you can go with 5% BSA.

apple:
Hi MaximinaNYC,

My primary ab is mouse monoclone ab. My cells are human leukemia cells.

Vector lab. suggests me use 0.1% BSA as a general blocking agent. What do you suggest?

Thanks

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