Ab94868

Human cerebral microvessel endothelial cells D3 cells were washed twice with cold phosphate-buffered saline (PBS) on ice, then the protein lysis buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4, 0.5% Triton X100, 0.5% sodium deoxycholate), and protease inhibitor cocktail (complete^®, Sigma) was added. The Bradford assay was applied to quantify the protein concentration (BSA as a standard). Total proteins were separated on a 7.5% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad), blocked for 2 h with 5% non-fat dry milk. Membranes were incubated overnight with a mouse anti-human TRPV2 primary antibody (1/250, sc-390439, Santa Cruz Biotechnology, Dallas, TX, United States), a polyclonal rabbit anti-human TRPV4 primary antibody (1/200, ab94868, Abcam) or a monoclonal mouse anti-human β-actin primary antibody (1/3000, Millipore). A secondary anti-mouse or anti-rabbit IgG conjugated to HRP (1/2000, Santa Cruz Biotechnology) was then incubated for detection using an ECL plus Western Blot Detection System (GE Healthcare, Velizy, France).

The expression of TRPV4, Bax and Bcl-2 was detected by immunohistochemical staining. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and then cut in 4 μm thickness. Immunohistochemical staining was performed using rabbit polyclonal anti-TRPV4 (ab94868; Abcam, Cambridge, UK), rabbit polyclonal anti-Bax (sc493; Santa Cruz, CA) and mouse monoclonal anti-Bcl-2 (sc7382; Santa Cruz, CA). After being washed three times with PBS, all sections were incubated in diaminobenzidine (DAB) reagents and counterstained with haematoxylin.

Frozen sections were rinsed in PBS 1X and permeabilized with PBS-0.05% Tx-100 solution for 30 min. Afterwards, to avoid non-specific staining, sections were incubated in a blocking solution with a 10% normal donkey serum (NDS, Jackson Immunoresearch, West Grove, PA, USA) during 1 h at room temperature. Then, the primary antibody rabbit anti-serotonin N-acetyltrasnferase (AANAT, ab3505, Abcam, Cambrigde, UK) was incubated at a 1:500 dilution at 4 °C overnight. Sections were washed in PBS1X-0.1% Tx-100 and incubated with donkey anti-immunoglobulin IgG rabbit antibody conjugated with fluorescein isothiocyanate (FITC; green, Jackson ImmunoResearch, West Grove) at 1:100 dilution in PBS-0.1% Tx-100 for 1 h in a dark chamber at a room temperature. The nuclei were stained with propidium iodide (red, Sigma-Aldrich, St. Louis, MO, USA) diluted 1:500 in PBS for 10 min. Finally, sections were rinsed and mounted in Vectashield (Vector Laboratories, Palex Medical, Barcelona, Spain) and coverslipped. The samples were examined under a confocal microscope (Zeiss LSM 5, Jena, Germany) at 40× magnification. For ciliary epithelial cells immunostaining, similar protocol was done for immunostaining of ciliary epithelium cells after incubating them with TRPV4 agonist GSK1016790A using a rabbit anti-TPRV4 (ab94868, 1:1000 Abcam) primary antibody.