Re: [Histonet] p16INK4a

From:Patti Loykasek

We have used several different clones of p16ink4. My understanding is the
polyclonal from Pharingen is no longer available. A really good clone out of
Europe is only available through Dakocytomation in their CINtek kit, this is
clone E6H4. We have tired the kit, and got good results. Currently we are
using the following:
Antibody: p16INK4a
Clone: 16PO4 (akaJC2)
Supplier: Lab Vision
Pretreatment: Steam for 40 in Tris, pH10
Titer: 1:200
Detection: Envision+
Localization: both nuclear & cytoplasmic

Hope this info gets you started. Good luck with your staining.

Patti Loykasek
PhenoPath Laboratories
Seattle, WA 




> The Pathologists I work for have requested that I purchase p16INK4a. I'd like
> to get an idea of which company most people order from and whether you use the
> monoclonal or polyclonal antibody.
> 
> Thanks,
> 
> Jean Taylor
> Immuno Tech
> GML
> Madison, WI  
> 
> -----Original Message-----
> From: Margaret Blount [mailto:mab70@medschl.cam.ac.uk]
> Sent: Wednesday, May 19, 2004 09:58
> To: 'Smith, Emily'; histonet@lists.utsouthwestern.edu
> Subject: RE: [Histonet] Background Staining
> 
> 
> Dear Emily,
> 
> That's a strange one. Have you titrated your secondary antibody
> sufficiently? what blocking solution do you use? Have the slides
> inadvertently dried out at any stage of the procedure?
> 
> These are a few thoughts that spring to mind. The other thing that I would
> look at would be to try charged slides from another supplier, or try
> Polysine or APES slides for comparison on indeed another batch of slides.
> That's my 2 penny worth - maybe someone else will have something more
> helpful to say and no doubt you have thought of some or all of these ideas
> yourself.
> 
> Good luck.
> 
> Regards
> 
> Margaret
> 
> Margaret Blount
> Chief Technician
> Clinical Biochemistry
> University of Cambridge
> Addenbrooke's Hospital
> Hills Road
> Cambridge
> CB2 2QR 
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Smith,
> Emily
> Sent: Wednesday, May 19, 2004 3:42 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Background Staining
> 
> 
> Hello All,
> In the course of a recent experiment, I saw strong background staining
> on charged slides with frozen cells in OCT (optimal cutting temperature)
> when treated with streptavidin-HRP.
> The staining appears over the entire glass surface not only where the
> cells and OCT were.  This all-over-staining also appears on clean
> (without OCT on them) slides that are processed with the OCT slides.  I
> have tried extensive water washes and the results remain the same.  No
> primary antibody is added and the results are the same.
> Have you seen this before? Do you have any suggestions?
> Thank you for your help.
> ~Emily
> 
> Emily A. Smith
> CuraGen Corporation
> 
> 
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