F4 80 monoclonal antibody

Immunofluorescence staining was performed as described before [24 (link)]. For double-staining monoclonal FAT/CD36 antibody was co-incubated with either polyclonal antibody directed against CK19, polyclonal antibody directed against F4/80 or monoclonal antibody directed against SMA. Cryosections of 5 µm thickness were fixed with acetone/methanol, washed in PBS and subsequently incubated with blocking medium (90% of a 0.1% BSA, 10% FCS in PBS) for 1 h at room temperature. The antibody dilutions were applied over night at 4 °C onto the sections using a FAT/CD36 monoclonal antibody from Abcam (Cambridge, UK) and F4/80 monoclonal antibody from Abcam (Cambridge, UK) CK19 polyclonal antibody from Abcam (Cambridge, UK) and SMA monoclonal antibody from Sigma-Aldrich (St. Louis, MO, USA) respectively. Non-immune serum was used as negative control. (4',6-diamidino-2-phenylindole (DAPI)); SouthernBiotech (Birmingham, AL, USA) served as nucleic acid stain. The slides were observed by using an Axiovert 200M epifluorescence microscope Zeiss (Jena, Germany).

Immunofluorescence staining was performed as described before 36 (link). Briefly, for double-staining monoclonal F4/80 antibody was co-incubated with polyclonal antibody directed against cytokeratin 19 (CK19), polyclonal antibody directed against MPO with monoclonal antibody directed against alpha smooth muscle actin (α-SMA). Cryosections of 5 μm thickness and cells were fixed with cold acetone/methanol, washed in PBS and subsequently incubated with blocking medium (90% of a 0.1% bovine serum albumin, 10% fetal calf serum in PBS) for 1 hr at room temperature. The antibody dilutions were applied over night at 4°C onto the sections using a MPO polyclonal antibody (Abcam, Cambridge, UK) and F4/80 monoclonal antibody (Abcam), CK19 polyclonal antibody (Abcam) and α-SMA monoclonal antibody (Sigma-Aldrich), respectively. Non-immune serum was used as negative control. DAPI (4′,6-diamidino-2-phenylindole; Southern Biotech) served as nucleic acid stain. The slides were observed using an Axiovert 200M epifluorescence microscope (Zeiss, Jena, Germany). The antibodies used for immunofluorescence staining and their concentration in this study are mentioned in Table 2.
The number of positive cells (MPO, F4/80) was counted at each time-point from total liver and in the portal areas from 10 different portal vessels in the liver.