[Histonet] Universal (protein) block for IHC

[Hermina Borgerink]

I routinely use casein in both my diluent as well as wash buffer for all
my immunos (I primarily use an alkaline phosphatase detection system,
but when doing double or triple immuno labeling I also use an HRP or
Beta Galactosidase system).  Before converting from the standard
protocol which used a non-immune serum from the same species as the
primary for blocking non-specific proteins, I ran a comparison and found
there to be no difference at all.  I use a Tris wash buffer that
contains 0.5% casein as well as 0.1% Tween 20 as a surfactant (I use the
MicroProbe system which employs capillary action).  My slides are clean
and have virtually no background staining.  The only time when I have
noticed some non-specific binding is when using goat primaries.  If
anyone would like my protocol, please respond privately since I cannot
attach on Histonet.

Hermina

Hermina M. Borgerink, BA, HTL(ASCP)QIHC
Wake Forest University Health Sciences
Department of Pathology
Medical Center Blvd.
Winston-Salem, NC 27157
Tel. (336) 716-1538
Fax. (336) 716-1515
e-mail hborgeri <@t> wfubmc.edu 
 
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Philip
Oshel
Sent: Thursday, April 07, 2005 11:01 AM
To: Histonet <@t> Pathology.swmed.edu
Subject: RE: [Histonet] Universal (protein) block for IHC

Kathy,

Casein is the main protein in milk. Skim milk has lots more stuff in 
it than just casein, and who knows what that's doing to your sections?

Phil

>I thought casein was just a fancy name for skim milk.  I use it
>sometimes as an avidin block by making a 5% solution out of cheap dried
>milk.
>
>Kathy
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kelly D
>Mcqueeney
>Sent: Wednesday, April 06, 2005 4:42 PM
>To: Johnson, Teri
>Cc: Histonet
>Subject: Re: [Histonet] Universal (protein) block for IHC
>
>Hi Teri,
>I noticed a decrease in specific staining when I plugged it into my
>protocol. I made it myself and it was a big pain. Buy it! Try
increasing
>
>antibody concentration on a few slides and see what happens. I never
use
>
>a block with my secondary.
>
>Good luck!
>
>Johnson, Teri wrote:
>
>>Has anybody converted to using a universal protein block (casein) for
>>their immunohistochemistry protocols rather than using species
specific
>>normal sera?  For those who have made the conversion, did you have to
>>make any changes to your methodology or did you just plug it in to
your
>>protocol and run?  Did you notice any difference in the staining
>>intensity for known antibodies (stronger, weaker, no change)?  Are you
>>also using it as a secondary antibody diluent or for washes?  Finally,
>>are you using commercially available reagent or are you making your
>own?
>>
>>Thanks so much!
>>
>>
>>Teri Johnson
>>Managing Director Histology Facility
>>Stowers Institute for Medical Research
>>1000 E. 50th St.
>>Kansas City, Missouri  64110
>>tjj <@t> stowers-institute.org
>>
>>
>>
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-- 
Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)

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