Orb76194

The cortex tissue of the left brain after 24 hours post-HI or cells treated in each group were centrifuged (1000 ×g, 15 min) to obtain the supernatants. After analyzing the concentration of protein, the protein sample was electrophoresed on SDS-PAGE and then transferred to the membrane. After blocking, add PIK3R2 (1:500, orb76194, Biorbyt, Cambridge, UK), LC3B (1:1,000; 2775S, Cell Signaling Technology, Danvers, MA, USA), Beclin1 (1:1,000; 3738s, Cell Signaling Technology, Danvers, MA, USA), p62 (1:500, orb89844), Cleaved-capase-3 (1:500, orb106556), Bax (1:500, orb224426), Bcl-2 (1:500, orb228150), β-actin (1:2000, orb178392) for incubation. p62, Cleaved-capase-3, Bax, Bcl-2 and β-actin are all from Biorbyt. Finally, it was incubated with goat anti-rabbit IgG antibody (1:1,000; ABIN101988) for 60 minutes. For 3-5 minutes, the membranes were seen using an ECL luminous substrate. Using ImagePro Plus, proteins were scanned, measured, and normalized to β-actin.

Thirty-four days post HI, the silences of cortex tissues of the left brain were administrated with dewaxing, and rehydration as usual. Then, they were removed using boiled citric acid buffer (BCA) for 8 minutes after being incubated with a 3% H 2 O 2 methanol solution for 20 minutes. The silences were blocked with 5% bovine serum albumin (BSA) for 18 minutes at room temperature before being incubated with PIK3R2 (1:300; orb76194; Biorbyt, China) antibody for 12 hours at 4°C. Afterward, the sections were rewarmed and incubated with a secondary antibody for 60 min. After visualization using DAB, the tissue specimens were counterstained, dehydrated, cleared, and fixed. Observe the slices under the microscope and count them.