TUNEL Staining and Microscopic Visualization of Apoptotic Cells

TUNEL staining was performed using the DeadEnd colorimetric TUNEL system (Promega, Tokyo, Japan) as previously described [12 (link)]. After 3,3′-diaminobenzidine (DAB) staining was used for visualization and detection of TUNEL positive cells, Nissl staining was performed and samples were observed using an optical microscope (BX-51; Olympus, Tokyo, Japan). Additionally, when streptavidin Alexa Fluor^TM 594 conjugate streptavidin (1:200; Life Technologies, NY, USA) was used for visualization and detection of TUNEL-positive cells, samples were counterstained for 10 min with 50 ng/ml of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Life Technologies, Carlsbad, CA) in PBS and observed using an inverted fluorescence microscope (BZ-X710; Keyence, Tokyo Japan) or a standard light microscope (BX51; Olympus) equipped with a DP72 digital imaging system (Olympus).