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Liver tissues were embedded in optimal cutting temperature (OCT) and 10 μm sections were cut and stored at −80°C till use. Frozen sections were fixed in ice-cold acetone for 5–10 min at room temperature. For double detection of CD24 and CK19 or single detection of either antibody, fixed sections were blocked with 10% mouse serum in phosphate-buffered saline (PBS) and then with a block for endogenous biotin using the Avidin/Biotin Blocking Kit (Vector Laboratories; SP-2001). For Fig. 3A Biotinylated monoclonal rat anti-mouse CD24 IgG2b antibody (BD Biosciences; 553260, 1:50) was incubated with sections overnight at 4°C and with Avidin D coupled to Texas Red (Vector Laboratories; A1100, 1:50) for 30 min at room temperature. Rat anti-mouse monoclonal anti-body CK19 IgG2a (TROMA III clone, a generous gift from Rolf Kemler at the Max-Planck Institute) (1:200) was subsequently incubated with sections. Fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat IgG2a secondary antibody (Serotec; MCA278F, 1:100) was used to detect CK19 specifically. For individual antibody staining of A6 and CD24 (Fig. 3B), biotinylated monoclonal rat antimouse CD24a IgG2b antibody (BD Biosciences; 553260, 1:50) was used and the secondary antibody for CD24 was FITC-conjugated donkey anti-rat polyclonal antibody (1:200). Rat anti-mouse A6 antibody (1:200) was incubated with sections. FITC-conjugated donkey antirat polyclonal secondary antibody was used to detect A6. Slides were mounted with coverslips using Vectashield mounting medium with DAPI (4, 6-diamidino-2-phenylindole) (Vector Laboratories; H-1200). All experiments included a negative control without primary antibody. The stained sections were examined using fluorescence microscopy.
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