[Histonet] immunohistochemistry

[Patsy Ruegg]

I have done extensive IHC work on bone and cartilage samples and in my
experience, controlled enzyme digestion such as pepsin at 37dc flat on a
slide warmer, or trypsin, or proteinase K are my first choices for AR on
bone samples.  Complete draining and airdrying is a must before heating the
slides, I, like Gayle try to airdry overnight after completely draining the
water by standing the slides up for at least an hour.  Drying them flat on a
slide warmer at 37-40dc is suggested.  I do heat them on the same flat slide
warmer (mine goes up to only 55dc) to melt the paraffin for maybe 30 min or
so.

Patsy 

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle callis
Sent: Friday, June 05, 2009 3:28 PM
To: 'Troutman, Kenneth A'; 'Histonet'
Subject: RE: [Histonet] immunohistochemistry

Good advice since actively boiling buffer puts mechanical stresses on
section, with bone more easily loosened by this action. It may help to
preheat the buffer to 95C before slide/section immersion into retrieval
buffer. A friend who is an IHC expert always did this, plus she used plastic
coplin jars (safer!).   

Also, we dry all our decalcified, paraffin embedded bone slides FLAT, at 37C
to 40C for overnight, with longer better - be sure slides are well drained
before laying them flat.  You can go to a 56C paraffin oven for a short time
(just enough to melt paraffin) after overnight if you desire, but you find
that unnecessary. 

Gayle M. Callis
HTL(ASCP)HT,MT
Bozeman MT 59715 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Troutman,
Kenneth A
Sent: Friday, June 05, 2009 3:03 PM
To: Histonet
Subject: Re: [Histonet] immunohistochemistry

I would recommend the following:
 
Citrate for 35 min at 95 deg.  Cool down for 10 min.  (This is our
protocol.)
 
Be sure you are using charged slides and I would let them air dry for at
least 1 hour before heating and deparaffinizing.  That might help, too.
Unfortunately, I don't think there is an effective method of retrieival that
will completely eliminate the possibilty of floating tissue.  
 
 Good luck.
 
Ashley Troutman BS, HT(ASCP)QIHC
Histopathology Laboratory
Department of Pathology
Vanderbilt University Medical Center
Nashville, TN
<http://www.vanderbilthealth.com/main/> <http://www.vanderbilt.edu/>
<http://www.mc.vanderbilt.edu/>  
Date: Thu, 4 Jun 2009 20:01:50 -0700 (PDT)
From: Hatem Salim <dr.hatemsaied <@t> yahoo.com>
Subject: [Histonet] immunohistochemistry
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <782588.78944.qm <@t> web46103.mail.sp1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

HI
 I am Research assistant at Physiology department of Michigan state
university. I am using the (ß-Catenin Antibody (Carboxy-terminal Antigen)
#9587 for IHC on mice femur sections. I have used this antigen unmasking
method: For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer
pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool
slides on bench top for 30 minutes.
The problem is that bone detachment always occurs so I wonder if there is a
method of retrieval that prevents bone detachment.
> Waiting to hear from you soon.
>  Thank you for your consideration
> Best wishes
> Hatem Salim
<http://www.vanderbilthealth.com/main/> 
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