Lc3ii

After the tissue paraffin sections were dewaxed, hydrated, and incubated in a hydrogen peroxide solution for 1 h at room temperature, LC3-Ⅱ (1:400, Abcam, Cambridge, UK) antibodies were used to evaluate autophagy in the injured spinal cord. The sections were observed and images were captured under a vertical fluorescence microscope (Olympus BX53, Tokyo, Japan). The sum of the area and the optical density (IOD) of LC3-Ⅱ was measured by ImageJ 5.0 software (Rawak Software Inc., Stuttgart, Germany). The average optical density of LC3-Ⅱ is equal to the total IOD divided by the total area. The above analysis was completed by three researchers who were blinded to the treatments.

Once the frozen hippocampal tissues were thawed, 10 μL RIPA was added to 1 mg of tissue, protease inhibitor cocktail, and PMSF, and then homogenized at a low speed with an electric homogenizer on ice until the tissue was completely lysed, the total protein was extracted and centrifuged at 12,000 r/min at 4 °C for 5 min, and this supernatant was removed. Protein quantification was tested with a BCA protein kit, then with SDS-PAGE gel, by sample loading, electrophoresis, mold transfer, Ponceau red to detect the presence or absence of bands, and TBST washing. Skimmed milk powder (5%) was blocked at room temperature for 2 h, and LC3Ⅱ and Beclin-1 (1:1000, Abcam, Cambridge, UK) antibodies were added dropwise overnight. The membrane was washed 3 times with TBST, 10 min/time, incubated with the secondary antibody on a shaker at room temperature for 2 h, washed with TBST again, identified by ECL chemiluminescence in the dark room, and then immersed in developer solution for 2 min and fixer solution for 2 min. ImageJ analyzed the gray value of the specific bands and compared the gray value of the target protein with β-actin as an internal reference.

The paraffin sections were routinely dewaxed, rehydrated, soaked in distilled water, dripped in a 3% hydrogen peroxide solution diluted with methanol, incubated at room temperature for 15 min, immersed in 0.01 M citric acid buffer, and then heated in a microwave oven at 98 °C for 22 min to repair. The sections were then washed with PBS 3 times, treated with rabbit anti-mouse LC3Ⅱ (1:1000, Abcam, Cambridge, UK), and stored overnight in a refrigerator at 4 °C. Then, the next day, the sections were washed 3 times with PBS, treated with goat anti-rabbit IgG/HRP polymer, placed in a humidified oven at 37 °C for 40 min, washed 3 more times with PBS, then developed with DAB for 10 min, and terminated with tap water. Once completed, the sections were dehydrated with gradient alcohol, made transparent with xylene, and sealed with neutral resin. Of the hippocampus tissue, 6 fields (×400) were randomly selected for each section to count the positive cells under high magnification and calculate the average value.