Agt 023

Paraffinized slides containing brain tissues were washed and incubated with IF-specific blocking solution (10% goat serum (AR0009, Boster, China) diluted in PBS) for 30 min. The slides were incubated with anti-GLUT3 (1:150, AGT-023, Alomone) overnight at 4 °C before being washed three times with PBS and then incubated with the Cy3 goat anti-rabbit antibody (Aspen). The slides were imaged using an Aperio VERSA 8 microscope (Leica, Germany), and the images were analysed using imaging software.
Neurons were fixed on coverslips with 4% paraformaldehyde for 30 min. After incubation with 0.5% Triton X-100 (Aspen) for 20 min, the coverslips were incubated in IF-specific blocking solution for 1 h at room temperature. Cells were incubated with anti-GLUT3 (1:150, AGT-023, Alomone) overnight at 4 °C. After the cells were washed with PBS, they were incubated with the Cy3 goat anti-rabbit antibody (Aspen) for 1 h at room temperature. The signal from the secondary antibody was captured using an IX73 fluorescence microscope (Olympus, Japan), and the images were analysed using imaging software.

Tissue and cellular proteins were extracted using RIPA total protein lysate (Aspen, China) following the manufacturer’s instructions. Proteins from each sample were separated by electrophoresis on 8–15% SDS-PAGE gels and transferred onto PVDF membranes (Aspen). After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight. After the blots were washed, they were incubated with HRP-conjugated secondary antibody for 1.5 h and detected by a chemiluminescent imaging system (Tanon, China).

To detect the surface expression of GLUT3, we harvested PC12 cells from 6-well plates using 0.05% trypsin 24 h after the third exposure to sevoflurane. The PC12 cells were washed twice with pre-chilled blocking solution and incubated with anti-(extracellular) GLUT3 (1:100, AGT-023, Alomone) at 4 °C for 1 h. After the cells were washed, they were incubated with phycoerythrin (PE)-Cy5 goat anti-rabbit IgG (Bioss, China) for 30 min. The samples were immediately subjected to flow cytometry on a CytoFLEX (Beckham Coulter, U.S.).
To detect the apoptosis rate of the PC12 cells, we analysed early and late apoptosis in cells by an Annexin V-PE Apoptosis Analysis Kit (AO2001-09A-G, Sungene Biotech) according to the manufacturer’s protocol. Briefly, PC12 cells were harvested, washed with cold PBS and then suspended in 1 × binding buffer. After the cells were resuspended, 100 µl of cells (1 × 105) was added to each labelled tube and incubated with 5 µl Annexin V-PE for 10 min. Then, 5 µl 7-AAD was added to each tube for 5 min. After PBS was added to a volume of 500 µl, the PC12 cells were subjected to flow cytometry.