In situ cell death detection kit

Brain tissues (n = 6) were put into 30% sucrose solution for 24 h and cut into 30 μm coronal sections using a Leica CM1950 cryostat (Leica Microsystems). After washing with PBS, coronal sections were sealed with 10% goat serum (C0265, Beyotime) and then incubated with primary antibodies of Drp1 (ab184247, Abcam) and Parkin (JF82-09, Novus). The sections were then sealed with 10% goat serum for 1 h and then incubated with Tomm20 antibody (H00009804-M01, Abnova). Thereafter, the sections were stained with fluorochrome-conjugated secondary antibody (ZF-0311, ZSGB-BIO), glial fibrillary acidic protein antibody (1 : 5000; ab7260, Abcam), and ionized calcium-binding adapter molecule 1 antibody (1 : 500; 019-19741, Wako). In brief, paraffin sections were immunostained with anti-nuclei (NeuN) antibody (ab177487, Abcam) at 4°C overnight and subsequently subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling staining using an In Situ Cell Death Detection kit (Cat. No. 11 684 795 910) according to the manufacturer's protocol. Finally, the sections were incubated with 4′,6-diamidino-2-phenylindole (C1002, Beyotime) nuclear dye, and the sections were visualized using a confocal laser scanning microscope (FV1000, Olympus). Sections from forebrain, midbrain, and hindbrain regions were randomly selected, and the ischemic penumbra was counted for statistical analysis.

For quantification of neuronal apoptosis at 3 days after TBI, double staining of neuron marker NeuN (red) and TUNEL (green) was conducted using the In Situ Cell Death Detection kit (Roche, South San Francisco, CA, USA), according to the manufacturer’s instructions. Coronal sections were counterstained with mouse anti-NeuN (1:1000, ab104224, Abcam, UK), at 4°C overnight and subsequently incubated with the In Situ Cell Death Detection kit and a secondary donkey anti-mouse Alexa 594 antibody for 1h at 37°C in the dark. The average number of TUNEL-positive neurons in the contused cortex was calculated by ImageJ software (Version 1.46r, Wayne Raband, USA).

For quantification of apoptotic cortical neurons, double staining of NeuN (red) and TUNEL (green) was performed using the In Situ Cell Death Detection kit (Roche, South San Francisco, CA, USA), according to the manufacturer’s instructions. Briefly, frozen sections were counterstained with mouse anti-NeuN (1:100, Abcam) at 4 °C overnight and subsequently incubated with the In Situ Cell Death Detection kit and a secondary donkey anti-mouse Alexa 594 antibody for 1 h at 37 °C in the dark. Finally, the sections were covered with DAPI and visualized under an inverted fluorescence microscope. The number of TUNEL-positive neurons was quantified manually in lesioned boundaries of six sections per brain at ×200 magnification using ImageJ software. Results were expressed as the apoptotic ratio of the total neurons (TUNEL-NeuN double positive stained cells/NeuN stained cells).