Quantitative Analysis of STIM1 Puncta

Immunofluorescence was performed as described elsewhere [31 (link)]. Cultured aortae or primary cultured VSMCs were fixed with 4% formaldehyde for overnight or 10 min respectively, followed by permeabilization with 0.1% Triton X-100 dissolved in PBS. The samples were blocked by 2% BSA at room temperature for 1 h before incubating with primary antibody at 4 °C overnight. After washing with PBS for three times, the samples were incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200) for 1 h at room temperature and mounted in 90% glycerol in PBS and the fluorescent signals were determined by a TCS SP5 confocal laser system (Leica, Germany). The 8-bit images were analyzed with ImageJ software [32 (link)]. Briefly, projected images were generated by collecting maximum pixel intensity of the in-focus frames into a single frame. The images were threshold to remove background fluorescence and used to create a mask image. Using the mask images, number of STIM1 puncta was scored with automatic “Analyze Particles” algorithm of ImageJ software and using cluster size of 3–100 pixels and circularity 0.1–1.0.