Antifade mounting medium

Immunofluorescence was used to detect the colocalization of LC3 and lysosomes. Cells were seeded in a 24-well plate and pre-treated with or without CQ for 2 h and then treated with COS for 48 h. After removing the medium, cells were fixed with methanol for 20 min on ice and rinsed with PBS 3 times. The cells were blocked with PBS containing 5% FBS and 1% Triton 100 and incubated with primary antibody at 4 °C overnight. After being washed 3 times with PBS, the cells were incubated with FITC-labeled (for LC3 detection) or Alexa Fluor 594-labeled (for lysosome detection) secondary antibody (1:50) (Yeasen, Shanghai, China) for 1 h at room temperature. An anti-fade mounting medium (Beyotime, Shanghai, China) was used to preserve the fluorescence signals. The fluorescence signals were detected using an inverted fluorescence microscope. The colocalization analysis was conducted through ImageJ software (ver. 1.52a, Wayne Rasband, NIH). Pearson correlation coefficient was used to represent the colocalization of red and green fluorescence signals.

Frozen coronal sections were used for detection of Cx43 and AQP4 with immunofluorescent. The sections were treated with buffer containing 0.2% Triton (Sigma) and 50 mM phosphate-buffered saline (PBS) for three times and 5 min each time, antigen retrieval buffer for three times and 5 min each time, 10% donkey serum for 1 h, and then polyclonal Cx43 (1:400; Abcam) or polyclonal AQP4 (1:400; Abcam) overnight at 4°C. After rinsed with PBS, the slides were incubated with DyLight 594 donkey anti-mouse secondary antibody (1:400; EarthOx), a kind of fluorophore-labeled donkey anti-mouse IgG (H+L) antibodies, for 1 h at 37°C. Sections were mounted with anti-fade mounting medium (Beyotime). Images were acquired using fluorescent microscope (Nikon) at a constant exposure.

The apoptosis of HeLa cells and HT-29 cells was detected using the Hoechst 33342 assay kit (Beyotime Institute of Biotechnology, China). HeLa cells (2 × 10⁵ cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the attached cells were washed with phosphate buffered saline (PBS) and fixed with freshly prepared 4 % paraformaldehyde for 30 min. After fixation, the cells were washed with PBS and incubated with Hoechst 33342 staining solution for 5 min. After staining, cells were washed with PBS and anti-fade mounting medium (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) was added, then the cells were viewed with a fluorescence microscope (Nikon Corporation, Tokyo, Japan). Apoptosis, as indicated by condensed and fragmented nuclei, was observed and recorded with the fluorescence microscope.
The HeLa cells (2 × 10⁵ cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the attached cells were washed with PBS and the DNA was isolated from HES-treated and control cells use DNA isolation kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China), separated by 1.0 % agarose gel electrophoresis, viewed and photographed by an ultraviolet light gel documentation system.

Mice were deeply anesthetized and perfused transcardially with ice-cold 4% paraformaldehyde (PFA) in PBS and brains were fixed in 4% PFA at 4 °C overnight. Frozen brain blocks were sliced into 20 μm sections using a cryostat (Leica Microsystems, Germany). The sections were subsequently washed for 15 min with 1.2% Triton X-100 in PBS, blocked in blocking buffer (5% normal goat serum, 2% BSA and 0.2% Triton X-100) for 1 h, and incubated with primary antibodies at 4 °C overnight. After washing with 0.1% Tween 20 in PBS, the sections were incubated with secondary antibodies for 2 h at room temperature. The sections were mounted with antifade mounting medium (Beyotime, China) and imaged using Zeiss Axio Imager A1 microscope (Carl Zeiss Microscopy GmbH, Germany). The primary antibodies used: rabbit-anti-GFAP (1:1,000, abcam, ab7260); rabbit-anti-IBA1 (1:1,000, abcam, ab178847). The secondary antibodies used: Alexa Fluor^® 488 Goat Anti-Rabbit IgG (H + L) Antibody (1:600, Invitrogen). The number and positive area of GFAP⁺ or IBA1⁺ cells were measured using the ImageJ analysis software.