Ab192878

Formaldehyde (4%)-fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C, and immunohistochemical staining was performed using a DISCOVERY XT automated staining platform (Ventana Medical Systems, Tucson, AZ, USA) with a DISCOVERY ChromoMap DAB (3,3’-diamniobenzidine) Kit as follows: Sections were deparaffinized and rehydrated using EZ Prep (Ventana) and washed with Reaction Buffer (Ventana), and antigens were retrieved by heating at 90 °C for 30 min in RiboCC citrate buffer reagent (pH 6.0; Ventana) prior to detection with an anti-Ki-67 antibody (ab15580, Abcam) or anti-NDUFB8 antibody (ab192878, Abcam) and DAB.

Animals were perfused with normal saline at a specific time point, and about 2 cm segments were cut from the injured spinal cord. Then, protein was isolated by homogenizing the segments after RIPA lysis. The protein concentration was obtained by the BCA assay. After electrophoresis, membrane transfer, blocking, the membrane was incubated with primary antibody overnight at 4°C. The following antibodies were used: anti-AMPK(1:2000,10929-2-AP, Proteintech, China), p-AMPK(1:2000, 2535T, Cell Signaling Technology, United States), anti-PGC-1α(1:1000, 66369-1-Ig, Proteintech, China),anti-TFAM(1:2000, ab252432, Abcam, United Kingdom), anti-Nrf1(1:2000, ab175932, Abcam, United Kingdom),anti-Sirt1(1:1000, 13161-1-AP, Proteintech, China),Anti-NDUFB8(Complex I) (1:2000, ab192878, Abcam, United Kingdom), Anti-SDHB(Complex Ⅱ) (1:2000, ab175225, Abcam, United Kingdom), Anti-UQCRC2 (Complex Ⅲ) (1:2000, abs116449, absin, China), Anti-COX IV (Complex IV) (1:2000, 4844s, Cell Signaling Technology, United States), Anti- ATP5F1A (Complex V) (1:2000, ab176569, Abcam, United Kingdom), anti-Beta Actin (1:5000, 66009-1-Ig,Proteintech, China). Membrane was incubated with the corresponding secondary antibody at room temperature for 1 h. Using ECL hypersensitive luminous solution to visualize the imprint and analyze the fluorescence intensity.

Protein preparation and immunoblotting methods were described in previous studies^{5 (link)}. The following antibodies were used: aggrecan (Millipore Sigma, AB1031, 1:1000), MMP13 (Abcam, ab39012, 1:1000), SIRT5 (Proteintech, 15122-1-AP, 1:500), TOM20 (Proteintech, 11802-1-AP, 1:1000), AIFM1 (Santa Cruz Biotechnology, sc-13116, 1:500), CHCHD4 (Proteintech, 21090-1-AP, 1:500), NDUFB8 (Complex I) (Abcam, ab192878, 1:2000), SDHB (Complex II) (Abcam, ab175225, 1:2000), UQCRC2 (Complex III) (Absin, abs116449, 1:2000), COX IV (Complex IV) (Cell Signaling Technology, 4844s, 1:2000), ATP5F1A (Complex V) (Abcam, ab176569, 1:2000), and succinylated lysine (PTM Bio, PTM-419, 1:500).