2.6. Immunocytochemistry Labeling and LipidTox Green Neutral Lipid Stain Analysis

MSCs, as well as day 0, 7, 14, and 28 differentiated cells, were fixed with a 10% neutral formaldehyde solution followed by permeabilization with 0.1% Triton X-100. Samples were blocked with 5% BSA in PBS for 30 min at room temperature prior to labeling with antibodies to the following: Cx43 (rabbit, 1:1000; Sigma-Aldrich; #C6219 St. Louis, MO, USA), perilipin (rabbit mAB, 1:100, Cell Signaling Technology #9349 Danvers, MA, USA). After washing, primary antibodies were followed by goat anti-rabbit secondary antibodies conjugated to Alexa 555 (1:1000; ThermoFisher; #A21425,) or goat anti-mouse secondary antibodies conjugated to Alexa 488 (1:1000; ThermoFisher; #A11070), and nuclei were stained with Hoechst (1:1000, ThermoFisher #H3570). Lipid accumulation was detected via LipidTox Green stain according to the manufacturer’s instructions (ThermoFisher #{"type":"entrez-nucleotide","attrs":{"text":"H34475","term_id":"979892","term_text":"H34475"}}H34475). Samples were imaged using a Zeiss LSM 800 confocal microscope equipped with a 63x/1.30 oil lens. Representative cell images reflect cell labeling performed at least three times on similar cell passages cultured on separate days.