Rabbit anti rat p erk1 2 monoclonal antibody

The RGC-5 cells grown on cover-slips were fixed in 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 20 min at 4°C. The cells were blocked in 5% BSA for 1 h at room temperature, followed by overnight incubation at 4°C with the primary antibodies: rabbit anti-rat GPR91 polyclonal antibody (1:200; NBP1-00861; Novus Biologicals LLC, Littleton, CO, USA), rabbit anti-rat p-ERK1/2 monoclonal antibody (1:200; #4370), rabbit anti-rat p-JNK monoclonal antibody (1:200; #4668) and rabbit anti-rat p-p38 monoclonal antibody (1:200; #4511; Cell Signaling Technology, Boston, MA, USA). The cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200; A21206; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.

Rabbit anti-rat pERK1/2 monoclonal antibody (1:100, # 4370) was purchased from Cell Signaling Tech. Co. Ltd., USA. Rabbit anti-rat ERK1/2 polyclonal antibody (1:100, BS-2637R) was provided by Bioss Biotech. Co. Ltd., Beijing, China. 3,3’-diaminobenzidine (DAB) and Power Vision two-step histo-staining reagent (PV-6001) were obtained from ZSGB-Bio. Co. Ltd., Beijing, China. Paraffin sections treated as above were de-waxed and washed according to routine procedures. The immunohistochemical procedures were performed in strict accordance with the manufacturer’s instructions. Under a microscope, the positive cells appeared with brown granules in the cytoplasm. Those sections to which 0.01 mmol/L PBS was added instead of primary antibody exhibited no positive responses. The positive cells were enumerated and averaged in five random views of cortex from four serial slices under a microscope at 400X magnification (Leica DMI400, Germany) The positive cell index (PCI = positive cells / total cells) was calculated and used to indicate the pERK1/2 and ERK expression levels.