I used to do BrdU staining on paraffin embedded mouse liver. (mice
were injected IP 1-2 hours before liver harvest with 50mg/kg BrdU) The
protocol we used is below. Most people say that Ki67 should be used
instead of PCNA. I have never tried either one.
1. Rehydrate through xylene and alcohols to water.
2. Boil in 10 mM Citric Acid pH 6.0. (Our microwave was set for 13
mins, but each microwave is different.) Allow slides to cool
afterwards for 10 mins.
3. Rinse in distilled water 10 mins.
4. Because liver is high in peroxidases, quench with 1.5% Hydrogen
Peroxide in Methanol for 10 minutes, and rinsed in running water for 5
5. Block endogenous biotin with avidin/biotin blocking kit from Vector
Laboratories(SP-2001). 15 mins in each solution at 37 degrees. followed
by PBS rinses.
6. Block slides with 4% Horse Serum in PBS either for 20 mins at 37
degrees or overnight at 4 degrees. (Usually the later since this
protocol takes so long to complete.)
7. BrdU mouse monoclonal from Roche/Boehringer Mannheim cat# 1170376
diluted at 1:500 in PBT (0.1% BSA, .2% TritonX-100 in PBS). Incubate 40
minutes at 37 degrees.
8. Wash in PBS twice for 5 minutes
9. Vector Laboratories biotinylated horse anti-mouse secondary (cat#
diluted 1:200 in PBT. Incubated 35 mins at 37 degrees.
10 Washed in PBS twice for 5 mins each.
11. Vector Laboratories VectastainABC Elite kit (PK-6100) prepared
during secondary antibody incubation because it must sit for 30 minutes
before using. Apply to rinsed slides and incubate 30 minutes at room
12. Vector Laboratories DAB kit (SK-4100) used. About 400ul per slide
for 5 minutes at room temperature..
13 Rinse in running distilled water for 5 minutes.
14 Counterstain if desired. Brief dip in Filtered Hematoxylin Stain,
Gill Formulation (Fisher Scientific?) followed by multiple water
15 Dehydrate through xylene and coverslip with Cytoseal 60.
> Would anyone care to share a BRDU & PCNA protocol?
> Also could you give source for these antibodies for rodent tissues.
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