Sqstm1 ab56416

ATG2A (#15011), ATG5 (#2630), ATG7 (#2631), BECN1 (#3738), BIRC2 (D5G9), CASP8 (1C12), FADD (#2782), FAS (C18C12), FASLG (#4273), HSP90 (C45G5); LAMP1 (C54H11), MLKL (D2I6N), PARP1 (46D11), RIPK1 (D94C12), RIPK3 (D4G2A), ULK1 (D9D7), and XIAP (3B6) antibodies were obtained from Cell Signaling. SQSTM1 (#ab56416) and STX17 (#ab116113) antibodies were purchased from Abcam. β-actin (ACTB; AC-74) and ATG2B (#HPA019665) were purchased from Sigma. MAP1LC3B (#NB100-2220) was purchased from Novus Biologicals. Cell lysis, co-immunoprecipitation, subcellular fractionation, and western blotting were performed as previously described^{19 (link),41 (link)}. Relative densities of the target bands to the reference bands was calculated using ImageJ (NIH).

All the specimens were serially sectioned to a thickness of 4 μm. After deparaffinization, the tissue sections were pretreated with 10 mmol/L of sodium citrate buffer (pH 6.0) in a microwave oven for 5 min at 95°C. The sections were then incubated in a mixture of 0.3% hydrogen peroxide solution in 100% methanol for 20 min to quench endogenous peroxidase activity. After washing with phosphate‐buffered saline, the sections were incubated overnight at 4°C with the primary antibodies for p62 (1:100; SQSTM1, ab56416; Abcam, Cambridge, UK). After incubation, the sections were rinsed with phosphate‐buffered saline and treated with the secondary antibody (Vector Laboratories, Burlingame, CA) for 30 min, followed by color development using 3,3′‐diaminobenzidine tetrahydrochloride (DAB; Cell Signaling Technology, Tokyo, Japan) to detect antigen–antibody binding, and hematoxylin was applied for 1 min to counterstain the nuclei. Epithelial cells from the plantar of mice were used as positive control. The samples stained without the primary antibody were used as negative control.