Rabbit anti p62 antibody

Hearts were dissected free from the surrounding connective tissue, and fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into slices using a microtome (Leica RM 2235 or Leica CM1850UV; Leica, Solms, Germany). The slices were then mounted onto glass slides, and histological examinations were performed. Immunohistochemistry was performed using Histofine Simple Stain kit (Nichirei, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, sections were deparaffinised with xylene and then rehydrated in a descending ethanol series. Sections were treated with 3% H₂O₂ in methanol for 15 min to inactivate endogenous peroxidases and then incubated with a primary antibody against p62 (rabbit anti-p62 antibody, 1:200; Proteintech); LC3 (rabbit anti-LC3 antibody, 1:200; Proteintech); CD68 (rabbit anti-CD68 antibody, 1:250; Abcam) at room temperature for 1 h. All sections were examined under an Olympus BX40 upright light microscope (Olympus, Tokyo, Japan).

Total protein was extracted in lysis buffer supplemented with protease and phosphatase inhibitors (Solarbio, Wuhan, China). Western blotting was carried out according to our previous publication (22 (link)), six zebrafish liver samples in each group were performed for Western bloting. The antibodies used were as follows: rabbit anti-GAPDH antibody (1:2000; servicebio), rabbit anti-COL1A1 antibody (1:1000; Wanleibio), rabbit anti-ACTA2 antibody (1:1000; Proteintech), rabbit anti-IL-1β antibody (1:2000; Wanleibio), mouse anti-IL10 antibody (1:1500; Proteintech), rabbit anti-DRP1 antibody (1:1500; Proteintech), rabbit anti-OPA1 antibody (1:1500; Proteintech), rabbit anti-MFN2 antibody (1:1500; Proteintech), rabbit anti-P-AMPK antibody (1:2000; Cell Signaling Technology), rabbit anti-AMPK antibody (1:1500; Proteintech), rabbit anti-PGC1α antibody (1:1000; Bioss), rabbit anti-NRF1 antibody (1:1000; Proteintech), rabbit anti-NRF2 antibody (1:2000; Proteintech), rabbit anti-TFAM antibody (1:2000; Proteintech), rabbit anti-PINK1antibody (1:2000; Proteintech), rabbit anti-PARKIN antibody (1:2000; Bioss) and rabbit anti-P62 antibody (1:2000; Proteintech). Protein expression level was normalized to that of GAPDH.

Radio immunoprecipitation assay buffer (P0013B; Beyotime, Shanghai, China) was used for the extraction of proteins from the liver tissue. Samples were resolved via SDS-PAGE on a 10% gel, and protein bands were delivered into polyvinylidene fluoride (PVDF) membranes. PVDF membranes were hindered in Tris-buffered saline with 0.1% Tween-20 containing 5% skimmed milk. The primary antibody diluent (P0023A; Beyotime) was used for incubation, and it was mildly stirred overnight at 4°C. Primary antibodies against p62 (rabbit anti-p62 antibody, 1 : 1000; Proteintech), IL-1β (rabbit anti-beclin 1 antibody, β-actin (anti-β-actin, 1 : 1000; Cell Signaling Technology), NLRP 3 (rabbit anti-beclin 1 antibody, 1 : 000; Proteintech), and beclin 1 (rabbit anti-beclin 1 antibody, 1 : 000; Proteintech, 1 : 000; Cell Signaling Technology) were utilized in this study. The secondary antibodies (anti-rabbit Ig-G, 1 : 1000) were utilized for incubation of the membranes for one hour. The analysis was conducted 3 times independently. The levels of protein were normalized using protein/β-actin ratios so that the differences of loading are minimized. The NIH ImageJ software was used for quantification of the relative signal intensity.