Sc 13177

Formalin-fixed, paraffin-embedded, and further dewaxed liver sections were used to evaluate the regional and cell-specific expressional pattern of MT₁ and MT₂ after 2 h of reperfusion. Using microwave irradiation, sections were exposed to antigen retrieval. By incubation in 3% H₂O₂-methanol, endogenous peroxidase activity was blocked. After subsequent treatment with normal rabbit serum, slides were incubated at 37 °C for 1 h with polyclonal goat antirat MT₁ or antimouse MT₂ primary antibodies (dilution 1:200; MT₁: sc-13186; MT₂: sc-13177; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A secondary antibody (biotinylated rabbit anti-goat antibody) was used for staining with streptavidine–biotin complex peroxidase. For chromogens, 3,3′-diaminobenzidine and 3% CoCl₂ were used, and slides were counterstained with hematoxylin.

Total cell extracts were prepared in 1× sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio, China). Cell proteins were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with GAPDH (1:1000, BM3876, Bosterbio, USA), p-PERK, p-IRE1, ATF4, P16 (1:500, bs-3330R, bs-16698R, bs-1531R, bs-20656R, Bioss, China), H3, NRF2 (1:500, D153567, D121053, Sangon Biotech, China), and TNF-α, IL-6, P21, MT1, MT2, HRD1, VCP, P65, p-P65, IKK (1:200, sc-52746, sc-32296, sc-136020, sc-13180, sc-13177, sc-293484, sc-136273, sc-514451, sc-166748, sc-7606, Santa Cruz, USA). Secondary anti-rabbit, anti-mouse antibodies (1:1000, BM2004, BA1001, Bosterbio, USA) conjugated with horseradish peroxidase were used. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence western blotting substrate (Thermo Scientific). Results were analyzed by Tanon-410 automatic gel imaging system (Shanghai Tianneng Corporation, China).