[Histonet] Re: Staining on Alcohol Fixed Smears

[Beth Cox]

I agree that with no formalin exposure, there is no crosslinking that 
needs to be broken, but a "then, do nothing" approach to antigen 
retrieval oversimplifies the issue.   Alcohol fixation makes changes in 
the protiens/antibodies also, although different changes than formalin 
does, and these changes need to be accounted for.  In my research on 
alcohol fixed smears, I found that most antibodies perform best with 
some antigen retrieval, often gentler than formalin tissues require.

Beth Cox, HTL/SCT(ASCP)QIHC
Vagabond Histotech

------------------------------

Message: 4
Date: Mon, 16 Dec 2013 11:13:59 -0500
From: Tom McNemar<TMcNemar <@t> lmhealth.org>
Subject: RE: [Histonet] Re: Staining on Alcohol Fixed Smears
To: 'Beth Cox'<bethcoxx <@t> gmail.com>,
	"histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>,"levi.fried <@t> gmail.com"
	<levi.fried <@t> gmail.com>
Message-ID:
	<E9A90E28259D2F4E84308C5E8EA8F7B4016951E94252 <@t> lmhs-exchange>
Content-Type: text/plain; charset="us-ascii"


Since there is no exposure to formalin, we do not do antigen retrieval on alcohol fixed specimens.  We use the Ventana Benchmark XT and copy the protocol to a new number and use the "Wet" option rather than paraffin.  No retrieval needed.

Tom McNemar, HT(ASCP)
Histology Co-ordinator
Licking Memorial Health Systems
(740) 348-4163
(740) 348-4166
tmcnemar <@t> lmhealth.org
www.LMHealth.org

-----Original Message-----
From:histonet-bounces <@t> lists.utsouthwestern.edu  [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Beth Cox
Sent: Saturday, December 14, 2013 6:30 PM
To:histonet <@t> lists.utsouthwestern.edu;levi.fried <@t> gmail.com
Subject: [Histonet] Re: Staining on Alcohol Fixed Smears

Levi,

I've worked up most of the more common antibodies on alcohol fixed
smears.  There are two things to remember here:  1) your specimens are
alcohol fixed, meaning antigen retrieval needs are different, and 2)
they are smears, meaning the cells weren't 'cut open' during microtomy,
which impeeds some nuclear staining.  Those are two different issues to
consider.

Some rules of thumb:
1)  most cell membrane antigens will stain nicely with minimal changes
to the protocols you use for FFPE specimens
2)  most cytokeratin antigens stain nicely with little change to your
FFPE protocols (if you need to change, start by cutting your antigen
retrieval time in half)
3)  most nuclear antigens require significant changes from FFPE
protocols, and some are essentially impossible to stain using standard
antibodies.

When I return to work on Monday, I will forward you my protocol
'adjustments' for the antibodies you requested.

Beth Cox, HTL/SCT(ASCP)QIHC
Vagabond Histotech




On 12/14/2013 12:00 PM,histonet-request <@t> lists.utsouthwestern.edu  wrote:

> Message: 4
> Date: Sat, 14 Dec 2013 18:25:08 +0200
> From: Levi Fried<levi.fried <@t> gmail.com>
> Subject: [Histonet] Staining on Alcohol Fixed Smears.
> To:histonet <@t> lists.utsouthwestern.edu
> Message-ID:
>        <CA+puqxTXL85pEwvR=sqPQA36tMJPm=sx2Kv3GYEbRbu0He3C2g <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi Everyone.
>
> I am looking to stain alcohol fixed smears with a group of antibodies.
>
> The antibodies of particular interest are p63, p53 and ki67.
> Along with these antibodies I am looking for positive nuclear and
> cytoplastic staining antibodies.
>
> If anyone has any experience in staining alcohol fixed slides your
> information is very appreciated.
>
> All the best.
>
> -- Sincerely, - Levi Fried