[Histonet] RE: Cleaved notch antibody/Trilogy

[Johnson, Teri]

Rachael, are you really using undiluted biotinylated goat anti-rabbit
and undiluted streptavidin-HRP from Zymed?  If so, that's your problem.
Those are concentrated reagents and are intended to be diluted before
use (from the data sheets I looked at, the concentration of the
secondary antibody is 0.75 mg/ml; the streptavidin is recommended to be
diluted at 1:150 - 1:750).

I'd start there and see if this helps.

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj <@t> stowers-institute.org



-----Original Message-----
From: Emerson, Rachael [mailto:Rachael_Emerson <@t> URMC.Rochester.edu] 
Sent: Wednesday, August 10, 2005 1:43 PM
To: Johnson, Teri
Subject: RE: Cleaved notch antibody/Trilogy


Hi Teri. Here is the protocol that I have been using.
It was derived from a lab looking at the facial features of embryonic
mice.

 <<Workbook1.xls>> 
Also note that I did some trials in the following manner and still saw a
ton of background:

Deparafinize & rehydrate to PBS
Trilogy vs Citrate Buffer vs. no antigen retrieval
Rinse in water, into PBS
Blocked with 3% H2O2 in MeOH
Rinsed with PBS
Incubated with Streptavidin-HRP for 30'
Rinsed in PBS
Developed in DAB

Any thoughts would be appreciated!
Thanks!!!!
Rachael Emerson


Rachael L. Emerson
Center for Pediatric Biomedical Research
University of Rochester Medical Center
575 Elmwood Avenue MRBX 1.11301
Rochester, NY 14642

Tel (585) 275-5073
Fax (585) 276-0232