RE: [Histonet] BrdU protocol on frozen brain tissue

From:"Danielle Crippen"

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 4C 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@buckinstitute.org


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Kelly D
Mcqueeney
Sent: Wednesday, March 23, 2005 6:06 AM
To: histonet@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

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