Rabbit anti trpv4

For immunofluorescence staining of serial brain coronal sections (12 μm) and cultured cells, the tissues and cells were blocked with 1% bovine serum albumin and 1% goat normal serum 1 h at room temperature. Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs). The sections and cells were incubated with following secondary antibodies: Alexa Fluor 555 goat anti-rabbit IgG (1:500; Invitrogen), Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen) for 1 h at room temperature. Then washed with PBS and stained with DAPI (4′, 6-diamidino-2-phenylindole).

Thioglycolate-elicited peritoneal macrophages were seeded in 10% FBS containing DMEM and incubated overnight. Unadhered cells were washed off, and 1% bovine serum albumin (BSA) containing DMEM was added to the plate and incubated for 3 h before ginkgetin treatment. To determine expression of CD36, TRPV4, TLR2, TLR4, TLR6, and GAPDH proteins whole cell lysates were prepared from cells treated overnight with ginkgetin (1 and 5 µM) or vehicle in the presence or absence of oxLDL (25 µg/ml). To determine the expression levels of LPS-triggered p-JNK and JNK, cells were pretreated with ginkgetin (5 and 10 µM) for 3 h and then stimulated with E. coli LPS for 30 min. Whole cell lysates were prepared from twice-washed cells (ice cold PBS) using RIPA buffer with added protease-inhibitor cocktail (Thermo Scientific, MA). Blots were probed with rabbit anti-TRPV4 (Alomone Lab, Jerusalem, Israel), anti-mouse CD36 (R&D, Minneapolis, MN), rabbit anti-TLR4, rabbit anti-TLR6, rabbit anti-JNK, and rabbit anti-p-JNK primary antibodies (Cell Signaling, Danvers, MA) followed by anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (R&D, Minneapolis, MN). Blots were stripped and re-probed with anti-GAPDH IgG (1:2000; Santa Cruz, Dallas, TX) for loading control.