Bx50 brightfield fluorescence microscope

Paraffin-embedded cochlear sections were dewaxed in xylene and rehydrated through a graded ethanol series and double-distilled water, followed by PBS washes. Positive control sections were incubated with 100 U/ml DNase I diluted in a buffer containing 20 mM Tris-HCl (pH 7.0), 10 mM MnCl_2, and 1 M NaCl at room temperature for 10 min. The TUNEL assay utilizing the In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland) was carried out following the instructions supplied by the manufacturer. Deparaffinized slides were incubated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity and then washed with PBS. As described in our previous report (Chen et al., 2018 (link)), the tissue sections were permeabilized first, blocked for 30 min at room temperature with the supplied blocking buffer, and then incubated with the TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. After PBS Tween-20 (PBST) washing, the tissues were stained with Converter-POD for an additional 30 min and washed with PBST. The diaminobenzidine chromogen was then applied for 10 min to label apoptotic cells. For histological analysis, the tissue sections were counterstained with hematoxylin and viewed with an Olympus BX50 brightfield/fluorescence microscope (Olympus Corp., Tokyo, Japan) equipped with a digital camera (Olympus DP74).

Cells (10⁴ cells/well) were seeded into six-well plates three days following lentivirus infection. The medium was replaced at two-day intervals. Following six days of culture at 37°C, cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. The fixed cells were then stained with freshly prepared diluted Giemsa (Merck KGaA, Darmstadt, Germany) for 10 min, washed with water and air-dried. The total number of colonies with >50 cells was counted using a Olympus BX50 Brightfield/Fluorescence microscope and an Olympus CH-2 Binocular Light microscope (Olympus Corp.).