Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488
(AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A
series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of
equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting
the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to
the number of proteins per mitochondrion. Under the normal condition, the measured mean copy
numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively