Matching fields for GFP images and X-gal images was the key issue to evaluate an individual cell for both GFP and X-gal. In order to match fields for GFP and X-gal staining, we put cross-striped scratches with a knife on the bottom of the plates before photographing for GFP. We photographed exactly matched fields for GFP and X-gal staining using
the cross-stripes as guides. Individual cells were identified morphologically and scored for positivity of GFP and X-gal staining. For liver sections, the livers were fixed with 4% neutral-buffered formalin, embedded in OCT compound, and sectioned at 6 μm. The sections were thawed in phosphate-buffered saline (PBS), photographed for GFP, reacted with X-gal, and photographed for X-gal staining. Fields were matched for GFP and X-gal based on the alignment of the sections. X-gal staining was performed using the β-gal staining kit (K1465-01, Invitrogen, Carlsbad, CA) according CP-690550 cost to the manufacturer’s protocol. The cells were fixed with 4% paraformaldehyde,
permeabilized with 0.05% Triton X-100, and blocked with Cytomation Protein Block Serum-Free (Dako, Glostrup, Denmark). The primary antibody against α-SMA was clone 1A4 (Dako) and that against FSP-112 was a kind gift PLX4032 from Dr. Eric G. Neilson (Vanderbilt University, Nashville, TN). The primary antibody for desmin (RB9014) was purchased from Lab Vision (Fremont, CA). The primary antibodies were incubated overnight at 1:200 for α-SMA and desmin and 1:300 for FSP-1, respectively. The cells were then incubated with the respective secondary antibodies conjugated with Alexa Fluor 594 (red) (Invitrogen). In case we needed to combine GFP fluorescence images and immunostaining, we photographed for GFP before staining, as GFP fluorescence is significantly attenuated after multiple washing steps of immunofluorescence. In order to match fields for GFP and immunofluorescence staining, we put scratches on the bottom of the plates before taking pictures for GFP. As β-gal activity is attenuated after multiple washing and
incubation steps of immunostaining, we performed check details X-gal staining first and immunostaining afterward. The cells were fixed with 4% paraformaldehyde and X-gal staining was performed. The cells were then permeabilized, blocked, and incubated with a primary antibody. The primary antibodies for α-SMA, FSP-1, and desmin were described in the previous section. The primary antibody for vimentin (JM-3634-100) was purchased from MBL International (Woburn, MA) and used at 1:200. Biotin-conjugated secondary antibodies and streptavidin-biotin complex/ horseradish peroxidase were bound. Diaminobenzidine was reacted to develop a brown color. The blue precipitation of X-gal staining (5-bromo-4-chloro-3-indolyl) was stable during immunostaining procedures.