WB
Western blot analysis of COX2/Cyclooxygenase 2 on different lysates with Rabbit anti-COX2/Cyclooxygenase 2 antibody at 1/1,000 dilution.
Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
Lane 2: C2C12 cell lysate (20 µg/Lane)
Lane 3: L6 cell lysate (20 µg/Lane)
Lane 4: Mouse bladder tissue lysate (40 µg/Lane)
Lane 5: Mouse small intestine tissue lysate (40 µg/Lane)
Exposure time: 3 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded rat bladder tissue with Rabbit anti-COX2/Cyclooxygenase 2 antibody at 1/100 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/100 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ICC/IF
Immunocytochemistry analysis of A549 cells labeling COX2/Cyclooxygenase 2 with Rabbit anti-COX2/Cyclooxygenase 2 antibody at 1/50 dilution.
Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-COX2/Cyclooxygenase 2 antibody at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.