Vectashield hardmount

Fluorescence immunohistochemistry was performed to confirm CTLA-4 expression levels in ex vivo lung cancer xenograft tissues. Tumor tissues were collected, frozen in Optimal Cutting Temperature compound (Sakura Finetek), and sectioned in 5 μm slices. For staining, slides were dried for 15 min at room temperature and fixed in 4% paraformaldehyde (Santa Cruz Biotechnologies), then washed twice with 1X PBS. Slides were then permeabilized in 0.2% Triton X (Sigma-Aldrich) in PBS for 15 min, washed with PBS, and blocked for 2 h in blocking solution at room temperature (5% donkey serum, 0.3% Triton X in PBS). Primary incubation with ipilimumab (25 μg/mL) in the blocking solution was performed overnight at 4°C. To reduce background, slides were then washed twice with 0.2% Triton X for 30 min and washed with PBS prior to incubation with goat anti-human 800CW secondary dye (LI-COR) for 1 h. Slides were subsequently washed twice with 0.2% Triton X for 20 min and twice with PBS for 5 min, mounted with VectaShield hard mount containing DAPI (Vector Laboratories) and coverslipped. Imaging was performed with a Nikon A1R confocal microscope.

Mice were deeply anesthetized by intraperitoneal injection of pentobarbital (100 mg/kg) and perfused transcardially with 20 mL of PBS, followed by 50 mL ice-cold 4% paraformaldehyde. The third to fifth cervical segments of the DRG, TG, or skin were removed, postfixed in the same fixative at 4°C, and placed in 30% sucrose solution for 48 hours at 4°C. DRG, TG (15 μm), or skin (30 μm) sections were incubated in blocking solution (3% normal goat or donkey serum) for 2 hours at room temperature and then incubated for 48 hours (DRG and TG) or 120 hours (skin) at 4°C with primary antibodies: anti-P2X3R (rabbit polyclonal, 1:4000; Chemicon (Temecula, Calif); guinea pig polyclonal, 1:12000; Neuromics, Edina, Minn), anti-GFP (chicken polyclonal, 1:20000; Aves Labs, Tigard, Ore), anti-RFP (rabbit polyclonal, 1:5000; MBL, Nagoya, Japan), and anti-TRPV1 (goat polyclonal, 1:2000; Neuromics). Following incubation, tissue sections were washed and incubated for 3 hours at room temperature in secondary antibody solution (Alexa Fluor 405, 488, or 546, 1:1000). The tissue sections were washed, slide mounted, and subsequently coverslipped with Vectashield Hardmount with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, Calif). Immunofluorescence images were obtained with confocal laser microscope (LSM510 or LSM700, Carl Zeiss, Thornwood, NY).