Re: IHC help please - brown everywhere

From:"J.L. Turnbull" <>

Dear All,

Thanks for all the help.  It's cleared up quite a few things.

I had done a pre-immune serum block before my primary antibody and i also
did an antibody dilution series (sorry - forgot to say).  I didn't think
of blocking for biotin though - thanks.

I did various antibody-free controls and put some slides straight into
developer - still brown.

I now suspect the fixative (my unfixed guts worked, but i couldn't use
them for microscopy) or endogenous peroxidase.

I'm still don't understand the chemical basis of the peroxidase quenching
step (which is driving me mad).  How can an enzyme be permanently quenched
by flooding with substrate which i then wash away?  I thought that enzymes
were recyclable?  How could it destroy the endogenous enzymes but not my
conjugated enzyme when i develop it? Is the activity non-enzyme based?
-that would then make sense?

Thanks so muchj for  all of your answers.  I've been getting lost in
thousands of different protocols for the past few weeks and then suddenly
got so many answers overnight!


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