Re: [Histonet] Antigen retrieval

From:rschoon (by way of histonet)


The following procedure has worked in my lab for years and has also been
published as it has been used in the publication of several articals and
reports.  If the directions are followed faithfully you will have
excellent results without nonspecific background.

Robert Schoonhoven

*_BrdU  Immunohistochemistry for use with Dako Envision^(TM) HRP Kit_*

*Antibody: anti BrdU*

Figure A: *Rat Kidney, 200x.*

Clone: Bu20a

Supplier:  Dako

Catalog No.: M 0744

Ig Species: Mouse

IHC Method: Formalin - Paraffin, Frozen, Plastic GMA & MMA

Pretreatment:  see method (below, 1-5)

IHC Handling: Manual

Isotype: IgG1

Procedure Species: all mammals

IgG Concentration: 1mg/ml

*_ _*

*_Specificity:  Proliferating Cells of all Mammals Treated with

* *

*_Staining Pattern: Nuclear _*

*_ _*


*_ _*

1) Hydrate slides to ddH_2 O as per SOP.

2) Hydrolyze with 4N HCl at 37^o C for 20 minutes.

3) Rinse with ddH_2 O for 1 minute at RT.

4) Transfer slides to a staining dish filled with ddH_2 O (kept at 37^o
C) for 5 minutes.

5) Incubate in pepsin solution (Dako) for 15 minutes at 37^o C.

*_All of the remaining steps are carried out at room temperature._*

6) Rinse 2X with ddH_2 O, 1 minute each rinse.

7) Rinse 2X with PBSt, 3 minutes each rinse

8) Peroxidase Blocking Reagent (Dako), 5 minute incubation.

9) Repeat step #7.

10) Primary antibody (anti-BrdU), incubate 10 minutes at a 1:200 dilution.

       [5 ?L antibody to 995 ?L 1% BSA in PBS]

11) Repeat step #7.

BrdU Page 1 of

12) Polymer labeled secondary antibody (Dako Envision HRP), incubate for
10 minutes.

13) Repeat step #7.

14) Incubate in working Dako DAB solution for 8 minutes (1 drop DAB
Chromogen per 1 ml substrate buffer).

15) Rinse well with  ddH_2 O.

16) DAB enhancer solution (2.5% CoCl_2 , 2.0% NiSO_4 (NH_4 )_2 SO_4 in
ddH_2 O) for 8 minutes

17) Repeat step #15.

18) Richard Allan Hematoxylin 1 for 25 seconds, rinse with tap water
until there is no blue color in the water.

19) Let sit in tap water for 5 minutes.

20) dehydrate and coverslip in accordance with SOP's.

/_Method developed by: Robert Schoonhoven_/

/_ _/

/_S.O.P. Effective January 13, 2003_/

          BrdU Page 2 of 2__

Steven Coakley wrote:

>Has anyone had problems with unwanted specific nuclear staining after
>performing heat AR especially from the microwave.  I've been attempting to
>stain paraffin embedded rat liver in which a 24hour and 48hour BDL with
>Brdu injection 1 hour prior to harvest.  With any form of AR, even limited
>80C I get 75-100% nuclear staining.  With no AR I get nothing, not even
>the bile ducts.  Does AR tend to effect nuclear staining with some
>antibodies.  I'm stumped.
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