A1r laser confocal microscope

Freshly-isolated split-open CD were fixed with 4% paraformaldehyde in PBS (pH = 7.4) for 15 min at RT. After fixation the samples were permeabilized by addition of 0.1% Triton X-100 in PBS for 10 min and washed in PBS 3 times for 5 min. Nonspecific staining was blocked with 10% normal goat serum (NGS, Jackson Immunoresearch, USA) in PBS for 30 min at RT. After washing with PBS (3 times for 5 min) the samples were incubated for 3 hr at room temperature in dark with anti-TRPV4 tagged with ATTO 550 (1:50, Alomone Labs, Israel; Cat. # ACC-034-AO) in 2% serum in PBS. After washing with PBS (3 times for 5 min) the samples were stained with DAPI (1.5 μM concentration, Calbiochem, San Diego, CA, USA) to visualize nuclei. Subsequently the samples were dehydrated, and mounted with permanent mounting medium (SouthernBiotech, Birmingham, AL, USA). Labeled tissue samples were examined with an inverted Nikon A1R confocal laser microscope using a 60X Plan-Fluor oil-immersion (1.3 NA) objective. Samples were excited with 405 and 561.7 nm laser diodes and emission captured with a 16-bit Cool SNAP HQ² camera (Photometrics, Tucson, AZ, USA) interfaced to a computer running Nikon NIS Elements 4.3 elements software. 3-D stacks of split-open CD were generated from series of confocal plane images with 0.25 μm step.

Standard wholemount immunohistochemical protocols were followed (see Vaasjo et al., 2018 (link); Beach et al., 2019 (link)). Tissues were dissected in normal saline (in mmol l⁻¹: NaCl 51.3, KCl 1.7, MgCl2 1.5, CaCl2 4.1, HEPES 5, pH 7.8.), and pinned in a Sylgard plate. Tissues were incubated in protease (0.5%; Type VI, Sigma; 10–15 min), washed thoroughly with normal saline, and then fixed in Zamboni’s fixative overnight (Mercedes Scientific, EKI 15395). Fixed tissues were washed 5 x 20 min in PTA (0.1 M phosphate buffer containing 2% Triton X-100 and 0.1% sodium azide) at room temperature. Following pre-incubation with normal goat or donkey serum (0.8%, 3–12 h, room temperature), tissues were transferred to the primary antibody (1:200 dilution in PTA, 3–5 days). Samples were washed (5 x 20 min in PTA) and incubated in secondary antibodies conjugated to a fluorescent marker (Alexa 488 goat anti-rabbit IgG (H+L) conjugate; Molecular Probes, Eugene OR) at dilutions ranging from 1:500 to 1: 1,000). Quality of the staining was assessed using a Nikon Eclipse fluorescence microscope prior to imaging. Confocal imaging was performed on a Nikon A1R Confocal Laser Microscope using the NIS Elements AR (Version. 4.5, Nikon Instruments). Stacks, z-series, overlays, and calibrations were obtained using the Fiji software (v. 2.00, NIH public domain).