BrdU Procedure

From:Robert Schoonhoven

This was also supposed to go out to the HistoNet but due to a multitude
of computer problems...............


What follows is my SOP for BrdU which has been published several times. 
My primary is purchased from DAKO,
PBSt is PBS containing 1.0% Tween 20, Aqua Hematoxylin and the DAB
Enhancer were purchased from 
Innovex but you could substitute if you wanted to.  With some minor
modifications it is easily automated.

BrdU Immunohistochemistry (DAKO Envision Kit)

1 - Hydrate slides to ddH2O as per SOP.
2 - Hydrolyze with 4N HCl at 37o C for 20 minutes.
3 - Rinse with ddH2O for 1 minute (at room temperature).
4 - Transfer slides to a staining dish filled with ddH2O (kept at 37o C)
    incubate for 5 minutes.
5 - Incubate in Pepsin solution (DAKO) for 15 minutes.

All of the following steps are carried out at room temperature.

6 - Rinse 2X with ddH2O, 1 minute each rinse.
7 - Rinse 2X with PBSt,   3 minutes each rinse.
8 - Blocking Reagent for a 5 minute incubation.
9 - Repeat step 7.
10- Primary antibody (anti-BrdU), incubate 10 minutes at a 1:200
       [5 mL antibody to 995 mL 1% BSA in PBS]
11- Repeat step 7
12- Polymer labeled secondary antibody, incubate for 10 minutes.
13- Repeat step 7.
14- Incubate in working DAB solution for 8 minutes (1 drop DAB per ml of 
    substrate buffer)
15- Rinse well with ddH2O
16- DAB Enhancer solution, 8 minutes.
17- Rinse well with ddH2O in staining dish.
18- Aqua Hematoxylin for 25 seconds and rinse with tap water until there
    no blue color to the water.
19- Place in tap water for 5 minutes.
20- Dehydrate and coverslip in accordance with SOP's.

Luis Chiriboga wrote:
> Hi all
> Has/is anyone doing any anti BRDU staining?  Can anyone recommended an
> antibody and a protocol? Has anyone tried it on a NEXUS?
> I am going to be staining mouse tissue (brain)
> Luis

best regards,
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
Chapel Hill, NC 27599
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123 

Don't go around saying the world owes you a living; the world owes you
nothing; it was here first. 
Mark Twain [Samuel Langhornne Clemens] (1835-1910)

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