Re: IHC help please - brown everywhere

From:"J. A. Kiernan" <>

On Thu, 19 Apr 2001, J.L. Turnbull wrote:

> caterpillar gut with a peroxidase-conjugated antibody. I've done quite
> a few runs and changed many variables - still brown.

> I fixed in 2.5% gluteraldehyde ...

This is why everything is brown. Free aldehyde groups of the
bound glutaraldehyde form covalent links to any protein
applied to the tissue, and that includes antibodies.

> Incubate in iodine/K iodide
This step makes no sense. It is for removal of precipitates
formed by fixatives that contain mercuric chloride.

> 1) the protcol said that the iodine step was to remove the sublimate, what
> sublimate?
Mercuric chloride (archaic name), which you didn't use.
> ... the other protocol used Hollandes fixative - would this make a
> difference.
It would probably be better in every way than glutaraldehyde,
especially when you're going to do paraffin sections. Apart
from its reactivity, glutaraldehyde does not go well with
paraffin embedding, and makes for difficult sectioning, It's
intended for specimens to be embedded in plastic. There are
reasons for all this, of course.

> primary monoclonal antibody in either milk. BSA, 
> Haemoglobin or pre-immune serum.  
A treatment with BSA _before_ the primary antibody is one of
the ways to try to block those free aldehyde groups of bound

> ... Washed many times.
It may not matter in your case, but excessive washing can
remove some bound antibodies.

A good introductory book about immunohistochemistry is
"Introduction to Immunohistochemistry" 2nd edn by JM Polak
and S Van Noorden. BIOS Scientific Publishers, 1997. (Royal
Microscopical Society Handbook #37) P'back, 141 pages.
Read the first 5 chapters before doing anything!            
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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