Mouse anti human rna pol 2 antibody

The HRECs were seeded on glass coverslips precoated with 5 mg/ml of fibronectin (Gibco) and allowed to grow to semiconfluence in a culture dish. The cells were washed with phosphate buffered saline (PBS) three times and fixed in fresh 4% paraformaldehyde (pH 7–8) for 10 min at room temperature. PBS was composed of 2.89 g of Na₂HPO₄·12H₂O, 8 g of NaCl, 0.2 g of KCl, 0.2 g KH₂PO₄ and 80 ml of ddH₂O. Next, the HRECs were permeabilized in 0.1% of Triton X-100 (Sigma, St. Louis, MO) for 5 min and blocked with 1% albumin from bovine serum (BSA; Sigma) in PBS containing 0.1% Tween 20 (blocking solution) for 60 min at room temperature. The cells were then incubated with rabbit anti-human heparanase antibody (1:300 dilution; Abcam, Cambridge, MA) and mouse anti-human RNA Pol II antibody (1:200 dilution; Abcam) overnight at 4 °C for the expression of heparanase and RNA Pol II, followed by incubation with appropriate secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 555 (1:200 dilution; Boster Biologic Technology, Ltd., Wuhan, China) for 2 h at room temperature. The cells were stained with 100 ng/ml of 4',6-diamidino-2-phenylindole (DAPI; Sigma) for 5 min, mounted with an antifading fluorescence medium (Vector Laboratories, Burlingame, CA), and imaged using a laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

The immunoprecipitated material from each sample was dissolved in sample buffer and boiled for 5 min before loading with SDS–polyacrylamide gel electrophoresis (SDS–PAGE) using a 10% Tris-glycine gel (Invitrogen, Paisley, UK) and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) at 250 mA for 90 min. Each membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 for 1 h at room temperature. The membranes were then incubated with polyclonal mouse anti-human heparanase antibody (1:800 dilution; Abcam) or mouse anti-human RNA Pol II antibody (1:200 dilution; Abcam) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature after washing with PBS containing 0.1% Tween 20. Finally, the blots were developed with a chemiluminescence reagent (Cell Signaling Technology Inc., Danvers, MA).