[Histonet] Help with MDM2 Protein Immunostaining

[Young Kwun]

Dear Gayle,

Thank you for your reply about the MDM2 staining.
I used citrate buffer in the Decloaker (a pressure cooker) for 5 min and
incubated with primary antibody overnight at room temperature and used a
polymer detection system (Ultravision) from LabVision for 30 min. I also
tried EDTA buffer and protease without much success.

With BondMax autostainer, I used pretreatment for 20 or 30 mininutes with
two different buffers (ER1-citrate based and ER2-high pH buffer) and ER2
gave much sharper nuclear staining. The primary incubation time was 30
minutes. The detection kit I used was Bond Polymer Refine Detection kit.

Thank you.







Young Kwun
Senior Hospital Scientist
Dept of Anatomical Pathology
Concord Hospital
Concord NSW 2139
Australia
Phone:61-2-9767 6075
Fax:61-2-9767 8427
Email:kwuny <@t> email.cs.nsw.gov.au
 

 

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: Friday, 1 June 2007 12:38 AM
To: Young Kwun
Subject: Re: [Histonet] Help with MDM2 Protein Immunostaining

If you tell us HOW you do your protocol, then people can help with your 
background staining - which can come from many sources?

At 05:53 PM 5/30/2007, you wrote:
>Dear Colleagues,
>
>
>
>Does anyone in Histoland use MDM2 Protein staining for some soft tissue
>sarcomas successfully?
>
>I tried Lab Vision's polyclonal antibody (1/100 dil) and Novocastra's MDM2
>(clone 1B10, 1/150 dil) with Decloaker HIER and BondMax Autostainer.
>
>However, my pathologist is not happy with the staining results, mainly
>because of some non-specific stains.
>
>Any help would be appreciated. Thank you.
>
>
>
>
>
>
>
>Young Kwun
>
>Senior Hospital Scientist
>
>Dept of Anatomical Pathology
>
>Concord Hospital
>
>Concord NSW 2139
>
>Australia
>
>Phone:61-2-9767 6075
>
>Fax:61-2-9767 8427
>
>Email:kwuny <@t> email.cs.nsw.gov.au
>
>
>
>
>
>
>
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>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610




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