Lsm800 meta laser scanning confocal microscope

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.

Mice were anesthetized and then perfused, cryopreserved, embedded, and sectioned as previously described (Liu et al., 2012 (link)). Immunohistochemistry was performed as previously described (Liu et al., 2012 (link)) with primary antibodies against trimethylated histone 3 lysine 9 (H3K9me3, 1:100; ab8898, Abcam), CC1 (1:100; OP80, Calbiochem), myelin basic protein (MBP, 1:500; SMI99, Covance), OLIG2 (1:200, ab81093, Abcam), NG2 (1:200; AB5320, EMD Millipore) or Caspr (1:100, ab34151, Abcam). Stained sections were visualized using confocal microscopy (LSM800 Meta confocal laser scanning microscope, Carl Zeiss Micro-Imaging). For NG2, CC1, OLIG2 cell counts, and H3K9me3 intensity quantifications, 4–6 20x fields were taken per mouse. For MBP-covered segments and internodal length marked by Caspr, 4–6 fields were taken per mouse followed by quantifications using ImageJ. One-way ANOVA tests were performed to assess statistical differences. For correlation of internodal length with social interaction ratio, data normality was determined using D’Agostino and Person test in GraphPad Prism 8. Pearson correlation coefficients were calculated if data passed normality test.