Af 488 conjugated secondary antibody

Immunofluorescence analysis was performed on MDA-MB-231 and MDA-MB468 cells to evaluate the co-localization Notch1 and mitochondria. Cells were grown on Lab-Tek chamber slides (Nunc) and then stained with a mitochondrion-selective probe (MitoTracker-Red, 200 nM) according to the manufacturer's protocol (Invitrogen). Following a PBS wash, cells were fixed in 2% PFA followed by blocking in 5% normal goat serum. Notch1 antibody (C-20, 1:50 dilution) (Santa Cruz Biotechnology) was added for overnight incubation at 4°C. Following PBS wash, AF-488 conjugated secondary antibody (1:200 dilution; Invitrogen) was for detection. Slides were then washed in PBS, mounted with DAPI (Vector Laboratories), and visualized using a Confocal Microscope (Olympus FV1000). The colocalization indexes Pearson's and Manders' coefficients were calculated using the ImageJ colocalization analysis plugin. The portions of the cell occupied by Notch1 and mitochondria were calculated as the ratio of the fluorochromes' areas (visualized by AlexaFluor488 and mito-Red, respectively) to the entire cell area (visualized by blue masking). After background subtraction, the mean intensity of each fluorescence was used as a threshold to obtain binary images on which the different areas were measured (n = 20 cells per group were analyzed).

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.