[Histonet] Re: Staining on Alcohol Fixed Smears

[Beth Cox]

I'm sorry, I have no experience with acetone fixed smears.

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

On 12/17/2013 8:40 AM, Susan Foreman wrote:
> What info do you guys have on acetone fixed smears?  Specifically, having
> trouble getting PAX5 (nuclear) to show good signal without chewing up the
> cells during antigen retrieval.
>
> Susan Foreman, HT (ASCP)
> KDL Pathology
> 315 Erin Drive
> Knoxville, TN 37919
> (865)584-1933
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tom McNemar
> Sent: Tuesday, December 17, 2013 5:48 AM
> To: 'Beth Cox'; histonet <@t> lists.utsouthwestern.edu
> Subject: RE: [Histonet] Re: Staining on Alcohol Fixed Smears
>
> Thanks for the clarification.  I appreciate the info!
>
> 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: Monday, December 16, 2013 2:25 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Re: Staining on Alcohol Fixed Smears
>
> 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
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