The Immunofluorescence assay was conducted as described in previous report (Zhou et al., 2015 (link)). In brief, the rats were anesthetized and transcardially perfused with a saline solution followed by 4% paraformaldehyde (Sinopharm Chemical Reagent Co. Ltd., Shanghai, P.R. China). The L4-6 DRGs were then removed and fixed in paraformaldehyde and dehydrated in 10, 20 and 30% sucrose (Sinopharm Chemical Reagent Co. Ltd.) in succession until sinking. The DRGs were cut at 14 μm thickness using freezing microtome (Leica, Wetzlar, Germany). The sections were incubated with blocking solution, and followed by the primary antibodies [anti-TRPV1 (Genetex, United States), anti-ASIC3 (Genetex, United States), anti-NeuN (Merck Millipore, Germany), anti-GS (Abcam, Cambridge, United Kingdom), anti-calcitonin gene-related peptide (CGRP) (Abcam, Cambridge, United Kingdom), anti-neurofilament (NF)-200 (Abcam, Cambridge, United Kingdom), anti-isolectin B4 (IB4) (Sigma, St. Louis, MO)] at 4°C overnight. After washing with PBS, the secondary antibodies labeled Alexa Fluor 488 and 555 (Molecular Probes, NY, United States) were incubated at room temperature for 1 h. The slides were observed under a fluorescence microscope, and the images were trimmed with AxioVision (Jena, Germany). Negative controls were performed by omitting the primary antibody.
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