Simultaneous Visualization of Astrocytes and Kir4.1

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).