Mouse anti cgrp

The proteins were separated by SDS-PAGE, which was preformed according to the instructions, and the separated proteins were transferred to a PVDF membrane. The membrane was blocked with skim milk. After washing with TBS-T, the membrane was incubated with the primary antibody at 4°C overnight. After thorough washing with TBS-T, the secondary antibody was applied to the membrane and detected using a chemiluminescence detection kit and related instruments. The primary antibodies used were mouse anti-CGRP (1 : 500, Santa Cruz Biotechnology, CA, USA), mouse anti-SP (1 : 500, Santa Cruz Biotechnology, CA, USA), anti-Oprk1 (1 : 500, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-p-P65 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-P65 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-p-IκBα (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-IκBα (1 : 1000, Cell Signaling Technology, Danvers, MA, USA).

The sections were deparaffinized for antigen retrieval and then blocked in BSA for 30 minutes at room temperature. The sections were incubated with the primary antibody overnight at 4°C. The slides were washed 3 times in PBS for 5 minutes each, and the secondary antibodies of the corresponding species were added dropwise and incubated for 50 minutes at room temperature in the dark. After washing 3 times with PBS, DAPI was added dropwise for nuclear staining and incubated for 10 minutes in the dark. After washing again, the slides were blocked. The primary antibodies used were mouse anti-CGRP (1 : 50, Santa Cruz Biotechnology, CA, USA), mouse anti-substance P (1 : 50, Santa Cruz Biotechnology, CA, USA), and anti-Oprk1 (1 : 50, Sigma-Aldrich, St. Louis, MO, USA).