[Histonet] Re: BrdU, Histonet Digest, Vol 16, Issue 37 17 & 22

[David A. Wright]

Hi Kelly, Danielle and the Histonet

RE: BrdU, Histonet Digest, Vol 16, Issue 37 
17. BrdU protocol on frozen brain tissue (Kelly D Mcqueeney)
22. RE: BrdU protocol on frozen brain tissue (Danielle 
Crippen) 

I routinely do BrdU detection on 40um sections of PFA-
perfused rat brain cut on a freezing sliding microtome.  We 
all like to stick with an antibody that works, but I switched 
to Chemicon's MAB3518 a while ago.  As it says in the product 
info, this doesn't require the harsh treatment with 2N HCl 
(and I also used to use Formamide/SSC  at 65C to separate the 
strands of the histone-stripped DNA for use with Sigma-
Aldrich's MAb).

Advantages:
     a) it's faster
     b) you use your normal protocol and reagents
     **there's much less background and tissue shrinkage.  
(If you see how nasty - white & opaque - the sections look 
after 30' in 2N HCl at 37C, and which doesn't all go away 
with the borate neutralization, you'll understand why).
     c) it's economical.  
Chemicon recommends 1:5000 -10,000 for acid alcohol fixed 
cells.  I use it at 10,000 (& 1:20,000 worked too!) overnight 
followed by Vector's biotinylated anti-mouse and ABC 
detection with Nickel & Cobalt intensification.

I have no connection with Chemicon other than being a loyal 
customer.

Good staining!
-David A. Wright
Section of Neurosurgery
University of Chicago

PS:  I routinely spin my Ab tubes on arrival and immediately 
aliquot for freezing at -80C.  Even with freshly calibrated 
pipettors, I find that there's always 5-10% less volume than 
the product info promises.  This is with several suppliers - 
Chemicon, AbCam etc.  Has anyone else noticed this annoying 
feature.  Maybe they hope people will use straight from the 
tube and not notice??? -DAW
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Message: 22 
Date: Wed, 23 Mar 2005 07:46:23 -0800 
From: "Danielle Crippen" <dcrippen <@t> buckinstitute.org> 
Subject: RE: [Histonet] BrdU protocol on frozen brain tissue 
To: <histonet <@t> lists.utsouthwestern.edu> 

Dear Kelly, 

We've had great success with the following protocol using the 
monoclonal BrdU Ab from Roche (1:100) on mouse brain tissue
(20um sections)and Vector's Avidin/Biotin blocking kit to 
block endogenous biotin: 

Immunocytochemical detection of BrdU-labled nuclei 
1. Air dry samples for 20min. 
2. cold MetOH fix for 20min @ -20C, air dry for 10min 
3. Then incubated at 37C (slide moat) for 30min in 2N HCl .. 
4. Sections are rinsed for 10 min at room temp in 0.1 M boric 
acid pH 8.5. 
5. Incubate the sections for 10 min 1x PBS. 
6. 15 min in 3% H2O2 in dH2O or MetOH 
7. wash in PBS 5 min 
8. Block in (1x PBS, 1% NHS, .1% BSA, .3% Triton) +Chicken 
anti MsIgG (2ug/ml) for 30 mins at RT 
9. Sections are then incubated overnight at 4?C in primary 
antibody diluted in block buffer (PBS with 1% horse serum, 
0.1% bovine serum albumin, and 0.3% Triton X-100) at its 
optimal concentration. 
10. After being washed in PBS 3x5min, sections are incubated 
in biotinylated secondary antibody (Vector)diluted in block 
buffer for 2 hr at room temperature. 
11. Make VECTASTAIN Elite ABC Reagent and allow VECTASTAIN 
Elite ABC Reagent to stand for about 30 minutes before use. 
12. After three 5 min rinses in PBS. 
13. Incubate sections for 30 minutes with VECTASTAIN ABC 
Reagent . 
14. Wash slides for 5 minutes in PBS. 
15. Incubate sections in peroxidase substrate solution (we 
use DAB kit from Vector) until desired stain intensity 
develops. 
16. Rinse sections in tap water. 
17. Dehydrate sections with 75%, 95%, 100% ethanol (3 min 
each) and Xylene (5 min). 
18. Mount the sections with permanent mounting medium. 


Danielle Crippen 
Morphology Core Manager 
Buck Institute for Age Research 
8001 Redwood Blvd. 
Novato, CA 94945 
415-209-2046 
dcrippen <@t> buckinstitute.org 

-----Original Message----- 
To: histonet <@t> lists.utsouthwestern.edu 
Subject: [Histonet] BrdU protocol on frozen brain tissue 


Does anyone have a protocol for BrdU IHC on frozen brain 
tissue? 

Thanks, 
Kelly McQueeney