RE: Immunostain for PCNA

From:"Demirs, John T."

My first reactions is not to use Ab from Santa Cruze. I have never found any of
their primaries to be any good. 
Having said that, We use overnight incubations at 4C, tittered out to
1:200-1:500. You should be doing Antigen Retrieval for nuclear staining and for
controls any tissue should have some level of cell proliferation some are better
then others i.e. liver, spleen, skin. 
John T. Demirs B. A. HTL (ASCP)
Wellman Laboratories of Photomedicine
Massachusetts General Hospital
Photopathology Dept. Edward 214
15 Fruit St.
Boston Ma 02114
617-726-6984 phone
617-726-1206 fax

> -----Original Message-----
> Sent:	Thursday, February 27, 2003 3:14 PM
> To:	HistoNet Server
> Subject:	Immunostain for PCNA
> Hi. I was wondering if anyone has any suggestions on a protocol for PCNA
> stain. I'm staining mouse lung tissue using the Envision+ kit from DAKO. I'm
> using a rabbit anti-PCNA from Santa Cruz biotech as the primary Ab. I incubate
> at RT for 1hr. Everything else is done as suggested by the kit (peroxidase
> block, serum block, secondary Ab, chromogen, etc). I used the PCNA Ab at 1:50
> and got nothing. When I went to 1:25, I had a lot of backrgound and no stain
> in the nucleus whatsoever. I'm wondering if anyone has any suggestions on this
> stain, and also what is the best tissue from mouse to use as a positive
> control. Thank you for your time.
> Nidal E Muvarak
> Associate Research Specialist
> Department of Biomedical Engineering
> University of Wisconsin-Madison
> 1550 Engineering Dr.; Rm. 2158
> Madison, WI 53706-1609
> Lab: (608) 265-8921; Office: (608) 265-4205; 
> Home: (608) 256-7934; Cell: (608) 332-6068

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