[Histonet] Re: BrdU, Histonet Digest, Vol 16, Issue
37 17 & 22
David A. Wright
dw18 <@t> uchicago.edu
Wed Mar 23 20:48:38 CST 2005
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
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-
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
-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
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>
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
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
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
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.
Morphology Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945
dcrippen <@t> buckinstitute.org
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
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