Airyscan confocal microscope

Total ROS was measured as previously described.^{35 (link)} In brief, BMDM were stimulated with PBS or 25 μg/ml OxLDL for 24 hours and then incubated with 10 μM 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA, Invitrogen, D399) for 20 min. Mitochondrial ROS was measured using MitoSox Red reagent according to the manufacturer’s protocol (Invitrogen). Cells were analyzed by Airyscan confocal microscope (Carl Zeiss) and FACS (BD Biosciences). Geometric means of FACS histograms were measured and presented as bar graphs.

Livers were removed from euthanized animals and fixed in 10% neutral buffered formalin in histology cassettes for 48 hours and then stored in 70% ethanol followed by paraffin embedding using standard procedures. For each embedded tissue, 4 μm thick cross sections were collected on microscope slides, which were subsequently used for immunohistochemistry staining procedures. The sections were deparaffinized in xylene and rehydrated in serial washes of ethanol. Antigen retrieval was performed in10 mM sodium citrate buffer pH 6.0 in a steamer for 30 min followed by cooling at RT for 20 min. Following antigen retrieval, tissue sections were permeabilized with 0.1% Triton X-100 in PBS for 10 min prior to blocking with 10% normal goat serum in PBS for one hour. Later, sections were incubated with CPS1 antibody (Abcam ab45956) at 1 μg/ml in the blocking buffer overnight at 4°C. After overnight incubation, the sections were stained with Goat anti-Rabbit Secondary Antibody (Invitrogen A-11012) for 1 hour at RT. The cell nuclei were counterstained with DAPI using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). The stained tissue sections were visualised with LSM 880 with Airyscan Confocal Microscope (Carl Zeiss, Oberkochen, Germany).