Fluorescent tunel staining

Chondrocytes were treated with miR-128a precursor, antisense oligonucleotide, and scrambled control and the number of apoptotic cells per 10⁵ cells in total was quantified using flow cytometry (BD Accuri C6, San Jose, CA) upon probing with Annexin-V-FITC Apoptosis Detection Kits (Enzo Life Sciences Inc., Farmingdale, NY). In a subset of experiments, apoptotic cells were confirmed using fluorescent TUNEL staining (Roche Diagnostics GmbH), according to the manufacturer’s instructions.

Fluorescent TUNEL staining (Roche Inc., Mannheim, Germany) was performed according to the manufacturer’s protocol and a previous study [40 (link)]. TUNEL staining was used to detect TXNIP localization with apoptotic cells in the hippocampus and cortex of SAH animals by using fluorescence microscopy (IX71, Olympus, Japan). TUNEL-positive cells in the ipsilateral basal cortex (left) in five different fields per animal were counted under microscopy under 400× magnification in a blinded manner. The number of TUNEL-positive cells/mm² predicted the severity of brain injury [44 (link)]. Meanwhile, negative controls were performed with labelling solution without the TUNEL reagent.