[Histonet] Confusing IHC results

Patsy Ruegg pruegg <@t> ihctech.net
Wed Jul 20 17:33:04 CDT 2005

In my experience the endogenous biotin in liver cannot be completely removed
even with the most aggressive AB blocking, this is why I do not use an
avidin/biotin detection system, I use labeled polymer instead.

Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
wk email pruegg <@t> ihctech.net
web site www.ihctech.net

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Trajkovic,
Sent: Wednesday, July 20, 2005 10:31 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Confusing IHC results

Well I have to tell you all that I am pleasantly surprised and elated that
so many of you have responded regarding my IHC situation.

Here is the additional info many of you have asked for:

I'm staining rat liver.

There are two protocols that have given me positive staining, with some non
specific staining as well. I was trying to clean up the nonspecific
staining, when I started doing the dilutions, and realized that it did not
matter weather dilution is 1:100 or 1:2000.

Slides were stained on Ventana Discovery XT, using DAB map kit, with heat. I
also ran the same protocol without heat and slides were slightly cleaner,
however, the signal was also weaker.

Protease 2 treatment for 4 min. 

Avidin / Biotin blocking 20 min

Serum free protein blocking from Dako

I use Dako diluent for the primary and the secondary. I also tried adding 1%
normal horse serum to the diluent. No change in the results.

I played around with primary incubation time (32 min - 1 hour), did not
alter the staining results. Longer (4 hours) incubation for the No heat
protocol yielded similar results.  


Vector universal- Elite kit (PK-6200) 1:200

I also tried these:

Vector- Rabbit anti Goat 1:200

Vector- Horse anti Goat 1:200

Santa Cruz - Bovine anti Goat. Tried 1:200, 1:400 and 1:600.

Jackson Immuno- Donkey anti Goat 1:200 and 1:400.


I also stained the same tissue on Dako autostainer, using Reveal, Borg and
EDTA epitope retrieval, without any success.


Here is another issue that surfaced during the writing of this email. That's
why it took me 2 hours to write it. 

I had to go in the lab and get some specific information, as well as start
some other IHC runs, when I came across information on my universal
biotinylated secondary.

I looked up the info in the Vector catalog for the ABC Elite kit PK-6200,
and the information says to be used with mouse or rabbit Ab's. Nothing about
goat Ab. It also says that this universal secondary should not be used on
rat tissue. None of this information seems to be in the kit insert. I had a
couple of people read it and we could not find it.  What is interesting, on
the slide I have two different sections of rat liver. One is normal and the
other is necrotic. Only the necrotic section is staining as described in
Toxicology and Applied Pharmacology publication (191 (2003) 211-226). The
other section has just slight non specific staining.

I hope I've given you enough info. I am now even more confused, but the
original deal is still on the table (or the bar).

Cheers, nazdrovije, ziveli, skol, ..... 


Dusko Trajkovic

PS. To ms. Rae Ann S. who responded with a, "Hey fella, you better watch out
what you are offering!!!! You can't just go 

around kissing anybody!!!! IT'S NOT ANYBODY. IT'S MY FELLOW HISTOTECHS. THE

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