Strategic distribution of the mitochondrial Ca2+ uniporter in cardiac mitochondria

αMCU (Sigma) was diluted either 1:50 in the range suggested by manufacturer (A) or 1:500 (as published by Lu et al. 2013, C). 2o antibody (ab) was conjugated with Alexa 488 (green fluorescence). Before fixation the cells were loaded with MtTr Red 50 nM (A,B) or 1 μM (as published by Lu et al. 2013 C,D). The images show the green (top) and red (middle) channels separately as well as overlaid (bottom). Controls were incubated with goat serum instead of 1o ab (B,D). Note the ‘patchy’ pattern for the MCU fluorescence signal at 1:50 ab dilution vs. the homogenous mitochondrial signal indistinguishable from that of MtTr at 1:500 dilution. Furthermore, the control with MtTr 50 nM shows no green fluorescence (B top), even though mitochondria still show up in the red channel (B middle) whereas at MtTr 1μM mitochondria still show up in the green channel (D top) with a perfect overlap with the pattern in the red channel (D middle and bottom).