in addition to using formaldehyde instead of the gluteraldehyde, I would also
incorporate an endogenous biotin blocking step (treat slides with diluted
avidin, wash, followed by a biotin solution) this will reduce the background
staining substantially.
Abizar
www.innogenex.com

"J. A. Kiernan" wrote:

> 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
> glutaraldehyde.
>
> > ... 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
>    kiernan@uwo.ca
>    http://publish.uwo.ca/~jkiernan