Re: BrdU Staining

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From:rschoonh@sph.unc.edu
To:bresee98@yahoo.com (Katie B), histonet@Pathology.swmed.edu (Histonet Server)
Reply-To:
Date:Thu, 29 Apr 1999 10:48:34 -0400 (Eastern Daylight Time)
Content-Type:TEXT/PLAIN; CHARSET=US-ASCII

Katie,

I do a lot of BrdU staining and image analysis.  While consulting on a
cell proliferation study (not here at UNC), there was a processing
problem (with the dehydrants) which resulted in decreased, faint and in
some cases no visable chromagen (DAB).  Also, we had a project here that
involved decalcification and we did not get the intencity of stianing
that I prefer for IA.  Both of these poblems were resolved by the
following methode which may be helpfull (or may not all things being
unequal).  I should note that we use the Dako anti BrdU and the Dako
Envision Kit.  Procedure follows.

BrdU Immunohistochemistry (using the DAKO Envision kit)

1  Hydrate slides to DDH2O as per S.O.P.

2  Hydrolyze with 4N HCL @ 37 oC for 20 minutes

3  Rinse with DDH2O 1X for 1 minute (at RT)

4  Transfer slides to DDH2O (kept at 37oC) for 5 minutes

5  Incubate in pepsin solution (Dako) at 37oC for 15 minutes

                   All of the remaining steps are performed at room
temperature

6  Rinse 2X with  DDH2O, 1 minute each rinse

7  Rinse 2X with PBSt, 3 minutes each rinse

8  Blocking Reagent (H2O2) - 5 minutes

9  Repeat step 7

10  Primary antibody (anti BrdU) incubate for 10 minutes; 1:200 dilution
(20 minute incubation        time for preputial gland only)

11  Repeat step 7

12 Polymer labeled secondary antibody - incubate for 10 minutes (15
minute incubation time for        preputial gland only)

13  Rinse well with DDH2O 

14  Incubate with working DAB solution (1 drop DAB per 1 ml buffer) for
8 minutes

15  Rinse well with DDH2O

13  DAB Enhancer solution (Innovex)- incubate for 5 minutes

14 Rinse well with DDH2O

15  Stain with Aqua Hematoxylin (Innovex)for 35 seconds and rinse with
tap water

16  Place in tap water for 5 minutes

Dehydrate and coverslip (we use Permount, Fisher) according to SOPís

The difference between this procedure and our normal one is the pepsin.


best regards,
Bob
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
CB#7400
Chapel Hill, NC 27599
Phone 
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123 

**Suppose you were an idiot... And suppose you were a member of Congress
...
But I repeat myself.-Mark Twain**

-- Begin original message --

> From: Katie B <bresee98@yahoo.com>
> Date: Wed, 28 Apr 1999 10:29:03 -0700 (PDT)
> Subject: BrdU Staining
> To: Histonet Server <histonet@Pathology.swmed.edu>
> 
> Histonetters:
> 
> I have been using BrdU labeling of cells for quite
> some time in my studies.  Lately, I've been having
> problems with the staining quality of the tissues from
> some animals.  I use BrdU from Sigma and anti-BrdU
> from Becton Dickinson.  Tissues collected are fixed
> with Zinc Formalin (Anatech) for a minimum of 48h,
> processed together, and stained in large batches using
> capillary gap immuno.  But about 10-15% of the animals
> don't stain for BrdU at all, not even in the positive
> control tissues (hair follicles, squamous epithelium).
> 
> It seems like the problem may be in the BrdU injection
> itself.  I even increased the concentration to 60mg/kg
> body weight for this last study, but still have this
> erratic staining results.  (Our protocol calls for
> 50mg/kg.)  I know the injections are done properly
> because I did them myself!
> 
> Injections were done by making a little "tent" of the
> abdomen musculature, by pulling upwards with forceps,
> while the animal is on their back, anesthestized with
> 4% halothaine.  This allowed me to inject into the
> body cavity and not hit any organs.  If I had hit the
> liver, would that account for the failure of the
> incorporation of the BrdU into other tissues?  I then
> wait 2h before sacrifice.
> 
> There just seems to be no pattern to this!  It's not
> associated with any type of study exposure.  Does
> anyone have any suggestions as to what else can
> contribute to this?  I'm wondering if trying antigen
> retrieval may help, but if the antigen isn't even
> there, what next!?
> 
> ===
> Catherine "Katie" Bresee Bennett
> Laboratory for Experimental Pathology
> Department of Veterinary Pathology
> Michigan State University
> 
> *new* e-mail: bresee98@yahoo.com
> 
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> 
> 

-- End original message --





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