Axiocam mrm monochrome ccd camera

PLA assay was performed with Duolink In Situ Orange Starter Kit Mouse/Rabbit (DUO92102-1KT, Sigma Aldrich) following the manufacturer’s recommendations. The primary antibodies used were rabbit anti-SMAD4 (ab40759, 1:100, Abcam) and mouse anti-HSPB5 (ADI-SPA-222, 1:100, Enzolife). Microscopy images were taken on an Axio Imager 2 (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were acquired using an AxioCam MRm monochrome CCD camera (Carl Zeiss GmbH) and analyzed using ImageJ software.

Cells on coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min at RT and incubated with 10 μM of Bodipy 493/503 (ThermoFisher Scientific) solution for 30 min. After washing, coverslips were mounted with ProlongTM diamond antifade mountant (Molecular Probes) containing 20 µM Hoechst (ThermoFisher Scientific). The detection of lipid droplets using Bodipy 493/503 was carried out using 470-nm excitation and 525-nm emission wavelengths. The staining was observed with Axio Imager 2 (Carl Zeiss Microscopy GmbH, Jena, Germany) connected to an Apotome 2 module (Carl Zeiss GmbH). Images were taken with a AxioCam MRm monochrome CCD camera (Carl Zeiss GmbH). The lipid-droplet number was determined by assessing the number of total lipid droplets and the cell number (minimum 100 cells per experimental condition) in the field for the calculation of the average lipid-droplet number per cell. In addition, a quantification of the average fluorescence intensity per cell was performed in the randomly recorded fields using imageJ software.

Cells were fixed with 4% PFA at 4 °C for 10 min and incubated in a solution of 3% BSA and 0.2% Saponin (Sigma Aldrich) for 20 min. Samples were incubated overnight at 4 °C with primary antibodies: anti-NLRP3 (Adipogen Cryo-2 or Abcam Ab4207) and anti-IL-1β (R&D systems, AF-401), anti-ASC (Cell Signaling Technology, B2W8U), anti-cathepsin B (Santa-Cruz S-12), anti-Caspase-1 (Adipogen, Casper-1) or anti-β-arrestin-2 (Ozyme, C16D9). Cells were incubated with the appropriate probes (anti-Rabbit PLUS, #DUO92002; anti-Goat MINUS, #DUO92006 or anti-Mouse MINUS, #DUO92004) for one hour at 37 °C. Probes were then ligated for 30 min at 37 °C, washed twice and amplified using the manufacturer’s polymerase for 100 min at 37 °C in the dark. Cover glasses were mounted with a drop of mounting medium containing DAPI (Invitrogen). Microscopy images were taken on an Axio Imager 2 (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Apotome.2 module (Carl Zeiss GmbH). Images were acquired using an AxioCam MRm monochrome CCD camera (Carl Zeiss GmbH) with filter sets 43 HE (Rhodamine/Alexa568) and 49 (DAPI).