4 6 diamidino 2 phenylindole (dapi)

Cortical neurons were harvested 24 hours post-OGD and washed 3 times with PBS, followed by fixation in 4% paraformaldehyde for 15 min. Afterwards, these cells were washed 3 times with PBS, 5 min/time. Neurons were permeabilized and blocked with 0.3% TritonX-100 and 5% goat serum at 37 °C for 30 min. Then, primary antibodies were added to incubate at 4 °C overnight (16–18 hours): P62 (rabbit, 1:200, 8420-1 AP, Proteintech), LC3B (rabbit, 1:200, 192890, Abcam), Cleaved-caspase3 (C-cas3, rabbit, 1:100, 9664 s, CST), and TUJ1 (mouse, 1:400, ab78078, Abcam). Subsequently, the samples were washed and incubated with secondary antibodies (Goat anti-rabbit IgG 647, 1:400, ab150079, Abcam; Goat anti-mouse IgG 488, 1:400, A23220, Abbkine) at room temperature for 3 hours. For TUNEL staining, samples were incubated at 37 °C for 1 hour. The secondary antibodies for TUNEL staining were prepared by mixing TdT enzyme and fluorescent labeling solution 647 in a 1:9 ratio. After PBS washes, DAPI (Beyotime, 1:3000) was applied for nuclear staining, and the anti-fluorescence quenching agent was used for sealing. Imaging was performed with a Leica confocal microscope, and ImageJ software (v.1.8.0) was used for measuring axon length and indicated positive neurons. Apoptosis rate (%) was calculated by counting the number of Tuj1⁺TUNEL⁺ or C-cas3⁺/Tuj1⁺ cells and normalizing them to the number of Tuj1⁺ cells (which marks total neuron count) in the same field of view. Autophagic neurons (%) were measured by counting the number of LC3B⁺/Tuj1⁺ or P62⁺/Tuj1⁺ cells and normalizing them to the number of Tuj1⁺ cells (which marks total neuron count) in the same field of view.