Sc 390572

Immunofluorescence was performed as described in previous reports.^{28 (link)} Briefly, L3-L4 DRGs were rapidly detached from mice perfused with 4% paraformaldehyde (BL539 A, Biosharp) and post-fixed with 4% paraformaldehyde for 2 h, followed by dehydration in 5%, 15%, 30% sucrose gradients, respectively, and sectioned at a thickness of 12 mm on a cryostat. After blocking at room temperature for 1 h and washing with PBST (containing 0.04% Triton), the slices were incubated with anti-rabbit ETS1 antibody (1:1000; A13302, ABclonal), mouse anti-glutamine synthetase (GS; 1:800; EM1902-39, Hua Bio), mouse anti-β-tubulin (1:800; M0805-8, Hua Bio), mouse anti-CGRP (1:50; SC57053, Santa Cruz), mouse anti-P2X3 (1:100; SC390572, Santa Cruz), or mouse anti-NF200 (1:100; 60,331-1-Ig, Proteintech) at 4°C overnight. After washing with PBST, slices were finally incubated with fluorescent-conjugated secondary antibodies Alexa Fluor^TM 488 goat anti-rabbit IgG (A21206, Molecular Probes) and Alexa Fluor™ 594 goat anti-mouse IgG (A10036, Molecular Probes). The sections were mounted on a mounting medium with DAPI (0100-20, Southern Biotech) and scanned using a high-resolution digital confocal microscope (Olympus FV1000, Japan).