Imagequant las 4000 min ccd camera

The frozen spinal cord L4–6 was ground and mixed with cold RIPA Buffer (200 µL per 20 mg of tissue) (Beyotime Biotech, SH, China). Forty micrograms of proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane using a minigel and mini transblot apparatus (Bio-Rad, Hercules, CA, USA). After being incubated in 5% nonfat milk containing 0.1% Tween-20 for 2 h, the membrane was infiltrated with the primary mouse anti-Syt-1 antibody (4 µg/mL, R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. The membrane was washed in Tris-Buffered saline Tween-20 (TBST) 3 times and incubated in the goat anti-mouse secondary antibody (1:3000, Servicebio, WH, China) at 37 °C for 1 h. GAPDH (1:1000, Servicebio, WH, China) was used as the protein control. The antigen-antibody complex was reacted with a horseradish peroxidase substrate (Millipore, Burlington, MA, USA) and visualized using the ImageQuant LAS 4000 min CCD camera (GE Healthcare, Boston, MA, USA). The bands were analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA). Values of Syt-1 were represented as the optical density ratio of the target protein bands to the related GAPDH bands.

Proteins were extracted from the grounded spinal cord segment using RIPA Lysate Buffer according to the manufacturer’s instruction (Beyotime Biotech, Nantong, China). The crude protein per sample was subjected to 10% SDA-PAGE and Western blotted on PVDF membrane using the Mini-PROTEIN Tetra Cell (Bio-Rad, Hercules, CA, USA). The membrane was incubated in blocking buffer with 5% nonfat milk for 2 h, and was subsequently immunolabeled overnight at 4 °C with rabbit anti-GLAST (1:500), anti-GLT-1 (1:500), anti-EAAC1 (1:500) or anti-β-actin (1:300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. The membrane was washed and treated with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (1:5000, Boster biotech, Wuhan, China) for 1 h at 4 °C. Visualization of the antigen-antibody complex was conducted with a horseradish peroxidase Substrate (Millipore, Billerica, MA, USA) using the ImageQuant LAS 4000 min CCD camera (GE Healthcare, Piscataway Township, NJ, USA). The bands were analyzed by Quantity One software (Bio-Rad). Beta-actin was used as the internal control. Values of GT were represented as the ratio of the optical density of the GT band to the density of the related β-actin band.