In Situ Cell Proliferation Kit, FLUOS

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- cells were fixed with 2% paraformaldehyde for 20 min and permeabilized with 1% Triton X-100 in PBS for 5 min. -	- Cells were labelled for 16h. - Cells were denatured with 2 N HCl for 30 min at 37°C, - Cells were blocked for 30 min by using 3% BSA in PBS.

Upstream tips
- cells were fixed with 2% paraformaldehyde for 20 min and permeabilized with 1% Triton X-100 in PBS for 5 min. -
Protocol tips
- Cells were labelled for 16h. - Cells were denatured with 2 N HCl for 30 min at 37°C, - Cells were blocked for 30 min by using 3% BSA in PBS.

Publication protocol

MPMECs transduced with either Ad-Cre or Ad-LacZ were grown on coverslips in EBM-2 medium containing 0.2% FBS for 24 h, followed by incubation with the same medium containing bromodeoxyuridine (BrdU) (10 ng ml−1) in the presence or absence of recombinant VEGF (20 ng ml−1) for an additional 16 h. The incorporation of BrdU into proliferating cells was detected by using the In Situ Cell Proliferation kit (Roche) according to the manufacturer's instructions. Briefly, cells were fixed with 2% paraformaldehyde for 20 min and permeabilized with 1% Triton X-100 in PBS for 5 min. DNA was denatured by incubation with 2 N HCl for 30 min at 37°C, followed by five rinses in PBS to bring the pH back to 7.0. Cells on coverslips were blocked for 30 min by using 3% bovine serum albumin (BSA) in PBS and then probed with a fluorochrome-conjugated anti-BrdU antibody (1:15; Roche) in a humidified chamber at 37°C for 60 min. After several washes with PBS, coverslips were mounted onto slides by using ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were taken by using a 40× objective on an Olympus inverted fluorescence microscope and processed by using the Cellsens Dimension software program. BrdU-positive cells were quantified by counting >200 cells per sample, and the proliferation index was calculated as the percentage of BrdU-positive nuclei/total nuclei.

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