Figure 1.

Colocalization of transfected stromal interaction molecule 1 (STIM1) or STIM2 with native STIMs identified by immunocytochemistry. Cells were transfected at 7 days in vitro (DIV) and fixed in PFA at 10 DIV. Secondary antibodies were Cy2 anti-rabbit and Cy5 anti-goat. Lasers and channels were distributed as follows: BFP (cell morphology marker) 405 nm (blue), Cy2 (STIM1, anti-rabbit) 458 nm, STIM2 + YFP 514 nm, STIM1 + mCherry 543 nm, Cy5 (STIM2, anti-goat) 633 nm. Overall, in the figure, STIM1 is shown in red and STIM2 in green. 3D-reconstructed Z-stacks, slow, high-resolution imaging mode, separate imaging tracks, and the GASP detector of Zeiss 880 were used for best dye/staining separation. Partial overlap of the transfected species with the immunocytochemically detected species was clear for STIM1 (A,D, respectively) and was different from those of STIM2 (B,E). It should be noted that some transfected STIM1&2 puncta (A) were not detected in the immunostaining for STIM1&2 (D), probably because the antibody detects fewer puncta in the fixed tissue, unlike the transfected species that is imaged in-toto. This can be seen in the merged images (C,F).