Bs 1353r

Brain tissue (5 μm) were cut and fixed in formalin buffer after being paraffin-embedded. Paraffin-embedded sections of brain tissue were first deparaffinized and dehydrated. Slides were incubated with 0.3% H₂O₂ for 10 min and washed with ddH₂O three times, 3 min each time. Antigen retrieval was undertaken in a pressure cooker for 10 min with 0.01 M citrate buffer and washed with phosphate-buffered saline (PBS) three times, 5 min each time. Five percent bovine serum albumin (BSA) was applied to the specimen sections to block nonspecific protein for 20 min at room temperature. The primary antibodies, which were rabbit antibodies against Beclin1 (bs-1353R, Bioss, China), Parkin (bs-23687R, Bioss, China), PINK1 (6946, CST, USA), or BNIP3L/NIX (12396, CST, USA), were incubated for 2 h at 37°C and washed with PBS three times, each time 3 min. After washing, slides were washed and then incubated with secondary antibody, goat anti-rabbit IgG (ab7090, Abcam, UK), at 37°C for 30 min and washed with PBS three times, 3 min each time. The sections were stained with diaminobenzidine for ~5–10 min. Five nonoverlapping visual fields at 400× magnification were analyzed from each sample using a Moticam 3000 (Hong Kong Special Administrative Region, China) microphotography system. The mean number of positive cells was calculated using Image-Pro Plus 6.0 software (Xue et al., 2014 (link)).

Total protein samples were obtained after lysing in RIPA lysis buffer (Yeasen, Shanghai, China). The concentration was examined by a BCA kit (Beyotime Biotechnology, Shanghai, China). The quantified protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Boston, MA, USA). After blocking in 5% skimmed milk, the membranes were probed with primary antibodies LC3II/I (bs-8878R, Bioss, Woburn, MA, USA), Beclin1 (bs-1353R, Bioss), ATG5 (bs-4005R, Bioss), ULK1 (bs-3602R, Bioss), p-mTOR (bs-3495R, Bioss), mTOR (bs-1992R, Bioss), p-P70S6K (#97,596, CST, MA, USA), P70S6K (#34,475, CST), NLRP3 (ab263899, Abcam), SOCS3 (bs-24250R, Bioss), TSG101 (bs-1365R, Bioss), CD63 (bs-23032R, Bioss), calnexin (bsm-52639R, Bioss), and β-actin (bs-0061R, Bioss) at 4 °C overnight, followed by application with Goat Anti-Rabbit IgG (ab672, Abcam) for 1 h. The membranes were developed using an enhanced chemiluminescence solution (Yeasen), and the band intensity was quantified by Image J software.

Cells treated as described earlier were washed three times with PBS and lysed in RIPA buffer with a 1% protease inhibitor. Cell lysates were centrifuged, and the protein concentration was measured using a BCA assay kit. Equal protein aliquots (10 μg) were fractionated by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 3% bovine serum albumin in TBST and incubated with primary antibodies of Bax (bs-0127R, Bioss, Beijing, China), p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), PCNA (bs-2006R, Bioss, Beijing, China), LC-3 (12741S, CST, Boston, United States), Atg-5 (10181-2-AP, Proteintech, Wuhan, China), Beclin-1 (bs-1353R, Bioss, Beijing, China), NF-κB (10745-1-AP, Proteintech, Wuhan, China), p-NF-κB (bs-0982R, Bioss, Beijing, China), STING (19851-1-AP, Proteintech, Wuhan, China), cGAS (ab252416, Abcam, Cambridge, United Kingdom), iNOS (ab15323, Abcam, Cambridge, United Kingdom), GBP5 (13220-1-AP, Proteintech, Wuhan, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), and GAPDH (PMK053C, BioPM, Wuhan, China) overnight at 4°C, and then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were developed using an ECL, and the films were exposed using a bio-imaging system (170-8265, Bio-Rad).