Rabbit anti trpv4

Human biopsy specimens and mouse tissues were homogenized in ice-cold hypotonic lysis buffer to obtain cytosolic extracts according to a method previously published by our group.⁶³ (link) Extracts underwent electrophoresis through a polyacrylamide minigel. Proteins were transferred onto a nitrocellulose membrane that was saturated with nonfat dry milk and then incubated with either rabbit anti-TRPV1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-TRPV4 (Novus Biological, Ltd, Cambridge, UK), rabbit anti-PPARα (Abcam, Cambridge, UK), or mouse anti–β-actin (Santa Cruz Biotechnology). Membranes then were incubated with the specific secondary antibodies conjugated to horseradish peroxidase (Dako, Milan, Italy). Immune complexes were shown by enhanced chemiluminescence detection reagents (Amersham Biosciences, Milan, Italy). Blots were analyzed by scanning densitometry (GS-700 imaging densitometer; Bio-Rad, Milan, Italy). Results were expressed as optical density (arbitrary units; mm²) and normalized on the expression of the housekeeping protein β-actin.

Mouse TG sections at 12 μm were blocked with 5% normal donkey serum (Jackson-ImmunoResearch) and incubated overnight with primary antibody: rabbit anti-TRPV4 (1:2500, Novus Biologicals). Immunodetection was accomplished with secondary antibodies (AlexaFluor-594, 1:600; Invitrogen). Images were acquired using Keyence Microscope (Keyence Co.). 4–6 sections/TG were analyzed. % of TRPV4-expressing TG neurons innervating the TMJ or masseter muscle (TRPV4 + FB/FB) was analyzed.