Lsm 700 zen

Immunofluorescence double staining of Kir4.1 with GFAP (a specific marker for astrocytes) was performed as published previously [14 (link),20 (link),29 (link)]. Briefly, fixed brain samples were embedded in paraffin and cut into four-μm thick sections. Sections were autoclaved for 20 min to retrieve the antigen, and blocked with 1% bovine serum albumin (BSA) for 30 min. The sections were incubated with primary antibodies for GFAP (mouse monoclonal, 1:50; Progen, Heidelberg, Germany) and Kir4.1 (rabbit polyclonal, 1:100; Alomone Labs, Jerusalem, Israel) at 4 °C overnight. Subsequently, secondary antibodies of tetramethylrhodamine-5- (and 6)-isothiocyanate (TRITC; red fluorescence) goat anti-mouse (1:50; Sigma-Aldrich, St. Louis, MO, USA), or fluorescein isothiocyanate (FITC; green fluorescence) goat anti-rabbit (1:50; Sigma-Aldrich) were respectively used for visualization. Immunofluorescence images were obtained with a confocal laser scanning microscope (Carl Zeiss Japan, LSM 700 ZEN, Tokyo, Japan).

Cells were grown on glass bottom dishes (MatTek Corporation, Ashland, MA, USA) and exposed to 100 μM of lansoprazole for 3 h. Cells were then washed with PBS and fixed in 4% (w/v) paraformaldehyde (PFA) in phosphate buffer, at room temperature. Fixed cells were then permeabilized in 0.1% Triton X-100. After blocking with 1% bovine serum albumin for 30 min at room temperature, cells were incubated with an anti-Nrf2 (H-300) antibody (1:50, sc-13032; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight, at 4°C. Following three washes in PBS, an Alexa Fluor 488 goat anti-rabbit IgG antibody (1:250; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added for 2 h, at room temperature. Finally, cells were stained with Hoechst 33342 dye (Dojindo Laboratories, Kumamoto, Japan). Images were obtained using a confocal microscope (Carl Zeiss Japan, LSM 700 ZEN, Tokyo, Japan). Three independent experiments were performed, and representative immunocytochemical analysis of the experiment is shown.

In some experiments, immunofluorescence double staining of Kir4.1 with GFAP was performed as published previously (Harada et al., 2013 (link); Nagao et al., 2013 (link)). Briefly, fixed brain samples were embedded in paraffin and cut into 4 μm thick sections. Sections were autoclaved for 20 min to retrieve the antigen, and blocked with 1% bovine serum albumin (BSA) for 30 min. The sections were incubated with primary antibodies for GFAP (1:50) and Kir4.1 (1:100) at 4°C overnight. Subsequently, they were incubated with secondary antibodies of tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC; red fluorescence) goat anti-mouse (1:50; Sigma-Aldrich) or fluorescein isothiocyanate (FITC; green fluorescence) goat anti-rabbit (1:50; Sigma-Aldrich), respectively, for visualization. Immunofluorescence images were obtained with a confocal laser scanning microscope (Carl Zeiss Japan, LSM 700 ZEN, Tokyo, Japan).