Total protein and cytosolic and nuclear protein in the ischemic cortex tissue were extracted with the corresponding protein extraction kit (Key GEN Biotech, China) following the manufacturer's protocols, and the protein quantity was assessed with the BCA protein assay kit (Key GEN Biotech, China). Equal protein quantities were separated using sulfate polyacrylamide gel electrophoresis (8%–12%), prior to transfer to polyvinylidene difluoride membranes (Millipore, USA). After that, they were blocked for 2 h in 5% milk before being exposed to HMGB1, TLR4, Bax, Bcl‐2, NF‐κB p65, Cleaved Caspase‐3, GAPDH and Lamin B antibodies for an entire night at 4°C. The blots were thrice rinsed for 10 min each, before 1 h incubation in matched horseradish peroxidase‐conjugated secondary antibody at room temperature 2 h. Following three more washes in TBST buffer, protein visualization was done on X‐ray film employing the Super ECL Plus Detection Reagent (Key GEN Biotech, China). Protein band quantification was done via Image J.
Significantly, during the operation, the ipsilateral ischemic cerebral cortex tissues were obtained, respectively, both were 1 cm3 in size. Each sample was divided into two parts. One part was stored in liquid nitrogen and prepared for protein and total RNA, whereas the other part was fixed in 4% buffered formalin and prepared for the histomorphological analysis.
Significantly, during the operation, the ipsilateral ischemic cerebral cortex tissues were obtained, respectively, both were 1 cm3 in size. Each sample was divided into two parts. One part was stored in liquid nitrogen and prepared for protein and total RNA, whereas the other part was fixed in 4% buffered formalin and prepared for the histomorphological analysis.