Rabbit anti phospho p65 ser536

The TLR7 agonist gardiquimod (Sigma) was dissolved in DMSO (Sigma) at a concentration of 100 μg/ml and stored in aliquots at −20°C. gardiquimod was added to cell cultures at a working concentration of 0.5 μg/ml. Antibodies for western blot include primary antibodies: rabbit anti-phospho-p65 (Ser536) (3033, Cell Signaling Technology), rabbit anti-p65 (4764, Cell Signaling Technology), rabbit anti-TLR7 (ab24184, Abcam), mouse anti-Lamp1 (H4A3, Santa Cruz Biotechnology), rabbit anti-IFN-α (YT5170, Immunoway), rabbit anti-GAPDH (AF1186, Beyotime) and secondary antibodies: HRP conjugated goat anti-rabbit IgG (H+L) (A0208, Beyotime), Alexa Fluor 594 goat anti-rabbit IgG (H+L) (A-11012, Thermo Fisher Scientific) and Alexa Fluor 488 goat anti-mouse IgG (H+L) (A-11017, Thermo Fisher Scientific). Antibodies for flow cytometry analysis were listed in Supplementary Table 1.

Rats (220–250 g) were anesthetized with i.p. 10% chloral hydrate at dose of 300 mg/kg, and perfused with 250 m L normal saline and 250 m L 4% ice-cold paraformalde (PFA) through the ascending aorta. The ipsilateral L4-L6 DRGs were collected, then post-fixed in 4% PFA overnight and dehydrated in 30% sucrose in PBS solution at 4 °C until it sank to the bottom. The DRG was sliced to longitudinal sections (16 μ m) and then were washed three times with PBS solution. They were blocked with 5% BSA for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C, following by being washed three times with PBS solution and incubated with secondary antibodies. For double immunohistofluorescence staining, the primary antibodies and secondary antibodies were mixed respectively. Primary antibodies used in previous experiments included rabbit anti-Nav1.7 (1:100, Merck Millipore, AB5390), rabbit anti-phospho-p65-Ser536 (1:100, Cell Signaling Technology, 3033), mouse anti-GFAP (1:200, abcam, ab10062), mouse anti-CGRP (1:200, abcam, ab81887), mouse anti-NF-200 (1:100, Boster, M05307–1) and IB-4(1:100, Sigma, L2895). The stained sections were posted on glass slides with a very fine brush and the images were captured through image J software by fluorescence microscope (Nikon TE 2000-E, Melville, NY).