[Histonet] brdu on ffpe rat fixed for up to 2 years in NBF

[Robert Schoonhoven]

Patsy,

If the animal was treated with BrdU initially, then yes I have.  Unfortunately All of my papers and reference materials are boxed up at the moment (preparing to move) but I have done this procedure so many times that I can pretty much reconstruct it from memory.


1) Hydrate slides to ddH2O as per SOP.
2) Hydrolyze with 4N HCl at 37oC for 20 minutes.
3) Rinse with ddH2O for 1 minute at RT.
4) Transfer slides to a staining dish filled with ddH2O
(kept at 37oC) for 5 minutes.
5) Incubate in pepsin solution (Dako) for 15 minutes at 37oC.

All of the remaining steps are at room temp.

6) Rinse 2X with ddH2O, 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 μL antibody
to 995 μL 1% BSA in PBS]
 
11) Repeat step #7.
                                                                                                         

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  ddH2O.
 
16) DAB enhancer solution
(2.5% CoCl2 , 2.0% NiSO4(NH4)2SO4 in ddH2O) 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.

Best of luck,

Bob Schoonhoven




________________________________
From: Patsy Ruegg <pruegg <@t> ihctech.net>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Friday, April 17, 2009 1:26:36 PM
Subject: [Histonet] brdu on ffpe rat fixed for up to 2 years in NBF

Anybody tried doing IHC for brdu on ffpe rat intestine and skin fixed for up
to 2 years in NBF?

Thank you,

Patsy



Patsy Ruegg, HT(ASCP)QIHC

IHCtech

12635 Montview Blvd. Ste.215

Aurora, CO 80045

720-859-4060

fax 720-859-4110

www.ihctech.net 

www.ihcrg.org



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