[Histonet] Confusing IHC results

[Patsy Ruegg]

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 


Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pruegg <@t> ihctech.net
web site www.ihctech.net
 

This email is confidential and intended solely for the use of the Person(s)
('the intended recipient') to whom it was addressed. Any views or opinions
presented are solely those of the author. It may contain information that is
privileged & confidential within the meaning of applicable law. Accordingly
any dissemination, distribution, copying, or other use of this message, or
any of its contents, by any person other than the intended recipient may
constitute a breach of civil or criminal law and is strictly prohibited. If
you are NOT the intended recipient please contact the sender and dispose of
this e-mail as soon as possible.


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Trajkovic,
Dusko
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.  

Secondaries:

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
BEST PEOPLE IN THE WORLD.



LEGAL NOTICE
Unless expressly stated otherwise, this message is confidential and may be
privileged. It is intended for the addressee(s) only. Access to this E-mail
by anyone else is unauthorized. If you are not an addressee, any disclosure
or copying of the contents of this E-mail or any action taken (or not taken)
in reliance on it is unauthorized and may be unlawful. If you are not an
addressee, please inform the sender immediately.
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet