[Histonet] Re: IHC background

[Johnson, Teri]

Louise,

You are using a protocol (including antibody concentrations) that is
optimized for use on formalin fixed tissues which have been TDE
decalcified, and it's not working well on alcohol fixed tissues
decalcified in formic acid/HCl. That's the problem.

Questions to ask:
1 - are you seeing any specific positive signal in the midst of the
background on the alcohol fixed samples?
2 - when you tried the Novolink kit, did you lose all staining in the
alcohol fixed samples, or also in your formalin fixed samples?

Some things to consider:
The antigen may not be conformationally preserved using alcohol. So,
it's possible the antibody does not recognize the alcohol-fixed protein,
but does recognize and bind with the formalin fixed one.

The decalcification with Formic/HCl may have additionally altered the
protein, and perhaps what you're looking for isn't even in there any
more!

Alcohol fixed tissues generally do not require any retrieval, since
alcohol does not cross-link or coagulate proteins. You might try using
the same detection method, but dilute out your primary antibody
concentration (use 2-3 serial dilutions from what you currently use) to
try to lower the background.

If you see no specific signal in any of your antibody stainings, you may
think about perhaps a different antibody (polyclonal vs monoclonal) that
may recognize the protein in alcohol fixed samples.

Good luck!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110


From: "louise renton" <louise.renton <@t> gmail.com>
Subject: [Histonet] IHC background
To: Histonet <@t> lists.utsouthwestern.edu
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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
"There are nights when the wolves are silent and only the moon howls".
George Carlin No trees were killed in the sending of this message.
However, many electrons were terribly inconvenienced.


Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110