Immunohistochemical Analysis of VDAC

Histological analyses were carried out as described previously (Niimi et al. 2018 (link); Zhu et al. 2017 (link)) with some modifications. The sections were washed with water after deparaffinization in xylene and dehydration in ethanol. The sections were incubated in 3% H₂O₂ diluted with phosphate buffered saline (PBS) for 15 min to remove endogenous peroxidase activity and incubated in 10 mM citrate buffer for 60 min at 90 °C for antigen retrieval. After three 5-min PBS washes, the sections were blocked in 10% goat serum (#426041, Nichirei Bioscience, Tokyo, Japan) for 10 min and incubated with a polyclonal rabbit anti-VDAC primary antibody (ab15895, Abcam, Cambridge, UK) at a dilution of 1:3000 overnight at 4 °C. Normal rabbit IgG (#2729, Cell Signaling, Beverly, MA, USA) diluted to the same concentration as a primary antibody was used as the negative control. After being washed three times for 5 min in PBS, the sections were incubated in Histofine Simple Stain Max PO (#414181, Nichirei Bioscience) as a secondary antibody at room temperature for 30 min. After three 5-min washes in PBS, the signals were developed using a diaminobenzidine (DAB) substrate solution kit (#425011, Nichirei Bioscience) and counterstained with hematoxylin.