Visualizing IRS-1 Colocalization with ER Tubules

Cells were seeded on coverslips in a six-well plate and transfected with various expression constructs for 24–36 h and then stained for immunofluorescence detection using confocal fluorescence microscopy or directly visualized for cells expressing GFP-tagged proteins as previously described^{87 (link)}. The images were collected with a 63 × 1.4 NA or 20× objective lens using appropriate laser excitation on a LSM800 Meta laser-scanning confocal microscope (Carl Zeiss). The detector gain was first optimized by sampling various regions of the coverslip and then fixed for each specified channel. Once set, the detector gain value was kept constant throughout the image acquisition process. Images were analyzed with Zeiss LSM Image Examiner Software. As previously described^{39 (link)}, colocalization between IRS-1 puncta and ER tubules was determined by calculating the Mander’s coefficient of the percentage of IRS-1 condensates overlapping with ER tubules.