Rabbit anti trpa1

The temporal bones were removed and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1.5 hr at 4°C. The temporal bones were decalcified by incubation in 10% EDTA at 4°C for 3–5 days. The EDTA solution was changed daily. The bones were then embedded in the OCT compound for cryostat sectioning. The sections of 10 μm thickness were washed in PBS, and nonspecific binding was blocked with 1% bovine serum albumin (BSA) and 10% goat serum in PBS plus 0.1% Triton X-100 (PBST) for 1 hr. The primary antibodies, chicken anti-Tuj1 (Abcam), mouse anti-myelin basic protein (Abcam), rabbit anti-Myo7a (Proteus Biosciences, Inc), mouse anti-Tuj1 (Abcam), rabbit anti-TRPA1 (Abcam), TRPC3 (Novus Biologicals), TRPC6 (Abcam), TRPV1 (Novus Biologicals), TRPV4 (Abcam), were incubated overnight at 4°C. After incubating the primary antibodies, the slides were washed three times with PBST and incubated with secondary antibodies for 1.5 hr at RT in the dark. We used Alexa Fluor 647-conjugated goat anti-mouse and Cy3-conjugated goat anti-chicken, Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 568-conjugated goat anti-mouse (Jackson ImmunoResearch Labs) in a dilution of 1:500. Other markers used were phalloidin-Fluor 647 (Abcam) for F-actin and DAPI (Sigma) for nuclear stain. The slides were then examined under a confocal microscope (LSM 510, Zeiss).

For immunofluorescence staining, the extracted DRG was fixed in 4% (w/v) paraformaldehyde at 4°C overnight. The samples were then embedded in OCT compound (Sakura Finetek Japan Co. Ltd., Tokyo, Japan) and frozen at -80°C. DRG sections (7 μm thick) were prepared on gelatin-coated glass slides using a cryocut microtome (Leica CM3050S, Wetzlar, Germany). The slides were blocked with 5% fetal bovine serum (HyClone) and 2% bovine serum albumin (MP Biomedicals, Santa Ana, CA, USA) in PBS for one hour at room temperature (RT), followed by incubation with rabbit anti-TRPA1 (1:200; Abcam plc., Cambridge, UK) at 4°C overnight. The tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC)-conjugated anti-rabbit secondary antibody (1:500; Vector Laboratories Inc., Burlingame, CA, USA) was incubated for one hour prior to visualization. The sections were mounted with Vectashield mounting medium (Vector Laboratories), and fluorescent images were obtained using a confocal microscope (LSM 5 Pascal, Carl Zeiss, Germany).

Mice were anesthetized with 10% chloral hydrate and perfused through the ascending aorta with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, the TGs were removed and post-fixed with 4% paraformaldehyde for 4 h. The samples were cut into 14-μm-thick frozen sections on a cryostat. The sections were incubated with primary antibodies (mouse anti-TRPV1, 1:1,000, Abcam; rabbit anti-TRPA1, 1:500, Abcam; rabbit anti-TRPM8, 1:500, Abcam; mouse anti-CGRP, 1:1,000, Abcam; mouse anti-NF200, 1:1,000, Abcam; mouse anti-IB4-FITC, 1:1,000, Sigma; rabbit anti-ZBTB20, 1:1,000, Atlas Antibodies AB; rat anti-ZBTB20, 1:2,000, Abcam) overnight at 4°C, followed by secondary antibodies (Alexa Fluor 555 donkey anti-rabbit IgG, 1:1,000 and Alexa Fluor 488-conjugated donkey anti-mouse IgG 1:1,000, Invitrogen) at room temperature for 2 h. The sections were then observed under an epifluorescence microscope. All images were made into figures using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA), with only minor adjustments to the contrast and brightness settings if necessary.