Anti pcna 10205 2 ap

Fixed tissues were dehydrated with ethanol and transparent with xylene, finally embedded in paraffin. The tissues were cut into cross‐section of a thickness of 5 μm. Following dewaxing and rehydration, the sections were incubated with a solution of 3% solution of hydrogen peroxide for 10 min to block endogenous peroxidase activity. Then, sections were incubated with a blocking solution (Catalog number: AR1010) for 30 min. Subsequently, tissue sections were incubated with anti‐CERCAM (16411–1‐AP, Proteintech) or anti‐PCNA (10205–2‐AP, Proteintech) overnight at 4°C in a moist chamber and then incubated with SV hypersensitive two‐step kit (Catalog number: SV0004, Boster). The sections were then stained with a DAB chromogenic kit and the sections were then sent for microscope analysis.

Anti-Parkin polyclonal antibody was generated by immunizing rabbit with GST-Parkin (residue 258-407) fusion protein expressed and purified from E. coli. Anti-PCNA (sc-56), anti-Myc (sc-40), anti-GFP (sc-8334) antibodies, and Protein A/G Plus-Agarose (sc-2003) were from Santa Cruz Biotechnology, Inc. Anti-PCNA (10205-2-AP) and anti-Myc (10828-1-AP) antibodies were from Proteintech Group, Inc. Anti-Parkin (CST4211) and anti-RPA32 (CST2208) antibodies were from Cell Signaling Technology. Monoclonal anti-Flag M2 antibody (F1804), anti-Flag M2 affinity gel (A2220) and Cytochalasin B (857777) were from Sigma-Aldrich. Anti-Rad18 (ab57447) antibody was from Abcam. Anti-Rad18 (A301-340A) was from Bethyl Laboratories, Inc. Anti-BrdU (347580) antibody was from BD Biosciences. Streptavidin Sepharose (17-5113-01) was from GE Healthcare. PEI (23966) was from Polyscienses, Inc. Lipofectamine® 2000 and Lipofectamine® RNAiMAX Transfection Reagents were from Invitrogen.

After fixing and permeabilizing, the tissues collected from the xenograft tumors were blocked with 1% bovine serum albumin in PBS for 30 min and incubated with anti-PCNA (10205-2-AP; Proteintech) and Alexa Fluor 488-labeled Goat Anti-Mouse IgG (H + L) (A0423; Beyotime Biotechnology) antibodies. DAPI staining was used to visualize cell nuclei. Visualization of positively stained cells was performed using a Leica DM2000 microscopy system (Leica Microsystems, Wetzlar, Germany).