[Histonet] Yeh glad to be back & ??

[Liz Chlipala]

Amy

I use cleaved caspase 3 from cell signaling technologies, it does require
HIER retrieval, which you can do on bone but you are going to have some
section loss.  I'll attach my protocol in an additional e-mail.  I run lots
of immunos on formic acid decalicifed bone samples with good success.  I try
to stick with enzyme retrieval either pronase or proteinase K.  Proteinase K
is my favorite.  What other antibodies are you looking at?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 735-5001
fax (303) 735-3540
liz <@t> premierlab.com
www.premierlab.com
 
Ship to Address:
 
Premier Laboratory, LLC
University of Colorado at Boulder
MCDB, Room A3B40
Boulder, CO 80309

-----Original Message-----
From: Amy Porter [mailto:portera <@t> msu.edu] 
Sent: Friday, September 22, 2006 2:07 PM
To: Patsy Ruegg; 'Liz Chlipala'; 'Histonet'
Subject: Re: [Histonet] Yeh glad to be back & ??

Thanks Patsy - Are you using Caspase 3 ?  or another Caspase for that  and 
is it active or cleaved - just curious.  I think I am going to have trouble 
all the way around.  We are trying to do immuno on mouse tibias that have 
been acid decalcified.  After several postings I am trying to look at 
alternative decalcification methods, I have been able to obtain some 
forelimbs from mice just to work with the decaling and processing.  Any 
pointers that you would have would be greatly appreciated.  Amy
----- Original Message ----- 
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
To: "'Liz Chlipala'" <liz <@t> premierlab.com>; "'Amy Porter'" <portera <@t> msu.edu>;

"'Histonet'" <histonet <@t> pathology.swmed.edu>
Sent: Friday, September 22, 2006 3:43 PM
Subject: RE: [Histonet] Yeh glad to be back & ??


> Amy,
> I agree with Liz on this, it is hard enough with Tunnel to distinguish
> between necrosis and apoptosis without adding decal.  I much prefer 
> caspase,
> you can count on the positive cells being just those under going 
> apoptosis.
> Patsy
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Liz 
> Chlipala
> Sent: Thursday, September 21, 2006 9:45 AM
> To: 'Amy Porter'; 'Histonet'
> Subject: RE: [Histonet] Yeh glad to be back & ??
>
> Amy
>
> I did this back a few years ago and found that the process of formic acid
> decalcification caused all the nuclei to be positive.  From what I 
> remember
> the use of formic acid is not compatible with the tunel technique.  Why
> don't you try using cleaved caspase 3 immunohistochemistry instead.
>
> Liz
>
> Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> Manager
> Premier Laboratory, LLC
> P.O. Box 18592
> Boulder, CO 80308
> phone (303) 735-5001
> fax (303) 735-3540
> liz <@t> premierlab.com
> www.premierlab.com
>
> Ship to Address:
>
> Premier Laboratory, LLC
> University of Colorado at Boulder
> MCDB, Room A3B40
> Boulder, CO 80309
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Amy Porter
> Sent: Thursday, September 21, 2006 7:09 AM
> To: Histonet
> Subject: [Histonet] Yeh glad to be back & ??
>
> Is anyone out there doing Tunel on FFPE acid decalcifed sections.  I am
> trying to stain for apoptotic cells using a Tunel kit on decalcified mouse
> femurs.  Not having much success, if anyone has any tips or do's / don'ts 
> I
> would really appreciate the information.  Thanks in advance,
>
>
> Amy S. Porter, HT(ASCP) QIHC - Supervisor
> Michigan State University
> Investigative HistoPathology Laboratory
> Department of Physiology / Division of Human Pathology
> 2100 Biomedical Physical Sciences Bldg. Room #2133
> East Lansing, MI  48824-3320
> Phone:  (517) 355-6475 ext 1480 / Fax:  (517) 432-1368
> Email:  portera <@t> msu.edu
> www.humanpathology.msu.edu
>
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