Mouse anti kca3

Frozen brain tissues were blocked with 10% goat serum in 0.01 M PBS for 1 h and then incubated overnight at 4 °C with primary antibodies (Abs). Brain sections (20 μm) were incubated with the following primary Abs: mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel) and rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The brain sections were then washed with 0.01 M PBS and incubated with the respective Alexa Fluor® 488- or 568-conjugated secondary Abs (1:500; Invitrogen Corporation). Fluorescent images were acquired using a TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). For imaging acquisition, a prescan of all samples was performed to ensure confocal settings below saturation. For each experiment, all images were obtained using the same confocal settings. Six slices at 120 μm intervals from each brain were used to examine Iba1-positive cells. Three microscopic fields (0.01 mm²) were randomly selected in each slice with the same reference position for quantification. The Iba1-positive cell number was counted in a blinded manner, and the area was measured by Leica LAS AF Lite software (Leica, Germany).

Total brain tissues were sonicated using a LabSonic homogenizer (B. Braun Biotech Inc., Allentown, PA, USA), and the protein concentration in the brain samples was then quantified using a bicinchoninic acid assay kit (Pierce, CA, USA). Samples were then analyzed by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% milk containing 0.05% Tween in PBS. The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel). Membranes were then probed with secondary horseradish peroxidase-conjugated antibodies (1:3000; Amersham Biosciences, Little Chalfont, UK) for 1 h at room temperature. The blots were then visualized using chemiluminescent peroxidase substrate (ECL prime; GE Healthcare). Immunoreactivity for each protein band intensity was quantified using NIH ImageJ software [24 (link)] and normalized to β-actin as a loading control.

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).