Alexa orange red 546 nm

Briefly, after 15 minutes fixation in 4% paraformaldehyde, cells were permeabilized for 10 minutes with PBS containing 0.2% BSA and 0.1% TritonX-100. Then, cells were incubated for 10 minutes with NH₄Cl in PBS to quench free aldehyde groups. Primary antibody was incubated overnight. After washing, cells were incubated with fluorochrome-conjugated secondary antibodies for 1 hour. Finally, slides were washed, incubated with the nuclear stain and mounted. Antibodies were as follow: antibody for the surface of intact mitochondria (1:300; MAB1273, Millipore); MCT-4 (1:100; Santa Cruz); secondary antibody for immunofluorescence was Alexa orange-red 546 nm (1:500; Invitrogen).

Antibodies were directed to Ki-67 (AB16667, Abcam), calponin (SC136987, Santa Cruz), BECN1 (sc-11427, Santa Cruz), LC3B (#3868, Cell Signaling), LAMP1 (sc-17768, Santa Cruz), F4/80 (ab6640, Abcam), CD11b (PA5-18727, Invitrogen), Sca1 (ab-51317, Abcam), Osteopontin (ab8488, Abcam), LC3A/B (ab58610, Abcam). Secondary antibodies for immunofluorescence were Alexa Green 488 nm and Alexa Orange-Red 546 nm (Invitrogen). 4,6-diamidino-2-phenylindole (DAPI) (D3571), Prolong Gold Anti-fade mounting reagent (P36930), Slow-Fade Anti-fade reagent (S2828). Osteopontin was obtained from SIGMA (Cat. # O2260).