Optical transparent glass bottom plates

Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). STING and cGAS immunohistochemistry was performed on paraffin-embedded tissue sections, using a polyclonal antibody against mouse/human STING raised in rabbit (NBP2-24683, 1:250, Novus Biologicals, Denver, CO, USA or 13657, 1:250, Cell Signaling Technology, Danvers, MA, USA) or against mouse/human cGAS raised in rabbit (201708-T10, 1:125, Sino Biological, Eschborn, Germany). For fluorescent microscopy of STING and cGAS, murine or human preadipocytes were grown on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) or glass coverslips and labeled with the same antibodies used for immunohistochemistry, and then visualized with AF488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Histology images were adjusted to equal white balance after acquisition. Flow cytometry analysis was used to detect STING⁺, cGAS⁺ macrophages and adipocytes, as described in [24 (link)]. Flow repository identifier of FACS data is FR-FCM-Z236.

Autophagosomes were labeled with Cell Meter^TM Autophagy Fluorescent Imaging Kit (AAT Bioquest, Sunnyvale, CA, USA) and photographed with an Olympus IX83 inverted fluorescent microscope. For a fluorimetric quantification of autophagy, we cultured cells in 96-well plates and stained them with a fluorescent Autophagy Assay Kit (MAK138-1KT, Merck), according to the manufacturer’s protocol. Autophagy-related gene product 5 (ATG5) and microtubule-associated protein 1A/1B-light chain 3 (LC3) were labeled with polylonal rabbit antibodies (Merck) in cells cultured on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany), or quantified with an in-cell ELISA (BioCat, Heidelberg, Germany). AF488-conjugated secondary antibodies were used for visualization. Lysosomes were labeled with Lyso Brite Orange (Bertin Bioreagent, Montigny le Bretonneux, France) and Lyso View 405 (Biotium, Inc., Fremont, CA, USA) in cells cultured on optical transparent glass-bottom plates.

For a fluorescent microscopy of mitochondrial content and morphology, preadipocytes were grown on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) or glass coverslips. Functional mitochondria were labeled with MitoTracker Red or its fixation-resistant substituent MitoBacon Orange (BioCat, Heidelberg, Germany). Mitochondria were also labeled with CellLight^TM Mitochondria-GFP BacMam 2.0 transfection system (LifeTechnologies Corporation, Eugene, OR, USA). Mitchondrial succinate dehydrogenase complex subunit A (SDH-A) and cytochrome c oxidase I (COX-I) level were measured with spectrophotometry (BioGnost enzyme cytochemistry kits) in cells cultured in 96-well plates, and SDH-A protein level was measured with ELISA (MyBioSource, Inc., Vancouver, BC, Canada).