Eclipse e 1000 microscope

Livers were formalin-fixed and 7-μm sections were routinely stained with H&E or a 0.1% Sirius Red-picric solution following standard procedures. The slices were examined with a Nikon Eclipse E-1000 microscope equipped with an Olympus DP72 camera. For collagen–fiber determination, a series of six random-selected fields from each slice were visualized and quantified using ImageJ software.

25.000 microglial cells from one adult mouse brain isolated as described in the “Ex vivo isolation of adult microglia” section were centrifuged for 5 min at 1000 rpm at RT using Shandon Cytospin 4 (Thermo Scientific) and collected in gelatinized slides. Cells were then fixed with 4% paraformaldehyde in PBS for 20 min at RT and washed three times in PBS. After blocking for 30 min at RT with PBS containing 1% BSA, 0.03% Triton and 10% normal donkey serum (Gibco), cells were incubated overnight at 4 °C with primary antibodies diluted in blocking solution. After washing in PBS, slides were incubated for 1 h at RT with secondary antibodies and DAPI (5 μg/mL) diluted in blocking solution. The primary antibodies were monoclonal mouse anti-C/EBPβ (1:1111, AbCam, ab-18336) and monoclonal rat anti-CD68 (1:1000, Serotec, MCA1957). The secondary antibodies were donkey anti-mouse Alexa 546 (1:1000, Molecular Probes, A21203) and goat anti-rat Alexa 488 (1:1000, Molecular Probes, A21208). Microscopy images were taken with a Nikon Eclipse E 1000 microscope and a digital camera Olympus DP72.