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After permeabilization with 0.1% Trinton X-100, the cells were incubated overnight at 4C with monoclonal anti-cytochrome C (1:200 dilution, Abcam), polyclonal anti-Bax antibody (1:50 dilution, Abcam) and polyclonal anti-cleaved caspase-9 antibody (1:50 dilution, Cell Signaling Technology) followed by incubation with a secondary donkey anti-goat Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) or donkey anti-rabbit Alexa Fluor 567 conjugate (Invitrogen) for 1 h at room temperature

All tests were performed between 06:30 a.m

its expression is the highest in alveolar type II cells