[Histonet] IHC background

[louise renton]

Hi all,

This may have been asked before, but i'm struggling to find reference to it.
I have a problem with high background when doing IHC on retrospective tisue
blocks.

The blocks (soft tissue, bone and hydroxyapatite implants)were fixed in
alcohol and decalcified in a home brew of formic acid  and HCl. The tissue
is primate, the antibodies anti human rabbit polyclonals.

I have optimised the antibodies to tissue from the same species (formalin
fixed and if necessary decalcified electrolytically in Sakura's TDE soln
normal overnight processing to wax). I have also run the antibodies on the
same TYPE of sample as the retro project (but one where the fixation and
decalcification was controlled by me) and got good clean crisp staining in
most of the right places. Some staining in serum in blood vessels but could
be excluded on morphology.

Brief method:
dewax
Quench in metghanol/H2o2
Normal horse serum block
primary overnight at 4 deg C
Pan secondary antibody (Vector) biotinylated
ABC kit detection/amplification
DAB

My problem is this: In the retro blocks there is HUGe baxckground, of a
muddy dark brown colour (instread of the well advertised golden brown
pigment) Everything stains, muscle, bone, bone cells, collagen.

Is there any way to retrieve the situation? BSA in the wash buffer?
Detergents?

I have tried using the Novolink kit acording to their protocol and lost all
staining
I have access to a Vector Elite kit but have not tried it yet

Any suggestions?. I am ready to tear my hair, kick the desk and cause
general mayhem by stealing everybody's left shoe.


with very best regards













-- 
Louise Renton
Bone Research Unit
University of the Witwatersrand
Johannesburg
South Africa
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George Carlin
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