Immunofluorescence Staining Protocol for Spinal Cord and DRG

Immunofluorescence staining was performed as previously described (Sun et al., 2017 (link)). Briefly, anesthetized mice were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Lumbar spinal cords (L3–L5) and DRGs were quickly removed and post-fixed in PFA overnight at 4°C. Then the tissues were transferred to 0.1 M phosphate buffer containing 30% sucrose at 4°C. The 40-μm-thick sections prepared by cryostat were washed 3 times and then blocked in 5% goat serum containing 0.3% Triton X-100 for 1 h at room temperature (RT). After incubation in the primary antibody overnight at 4°C, sections were incubated with the secondary antibodies at RT for 2 h and then were counterstained with DAPI. For primary antibodies, we used rabbit anti-CGRP (1:500, ImmunoStar, United States), Isolectin IB₄ Conjugate (1:500, Invitrogen, United States), mouse anti-PKC-γ (1:200, Santa Cruz Biotechnology, United States), mouse mouse anti-PV (1:2000, Sigma-Aldrich, United States), rabbit anti-VGAT (1:500, Millipore, United States); For secondary antibodies, we used Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse (1:500, Abways Technology, China). The Fluorescence images were captured using a laser scanning confocal microscope (Nikon A1 Confocal System, Japan).