Whole-mount immunofluorescence staining of cell-loaded scaffolds

Whole-mount IF staining of the cell-loaded scaffolds was performed as described previously (Wang et al., 2010 (link)). In brief, the samples were collected at the indicated time points and fixed in 4% paraformaldehyde for 12 h. After thoroughly washing with PBS solution, the scaffolds underwent sequential membrane permeabilization and blocking with nonspecific antigens. Subsequently, they were incubated overnight at 4°C using the following primary antibodies: mouse anti-human ALB (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-human CYP3A4 (dilution, 1:100; Proteintech, China), rabbit anti-human MRP₂ (dilution, 1:50; Proteintech), and Alexa Fluor^®647 Mouse anti-Human CD31 (dilution, 1:100; BD Biosciences). Samples not incubated with specific primary antibodies served as negative controls. Next, cell-loaded scaffolds were incubated with FITC-conjugated Goat anti-Rabbit IgG Secondary Antibody or FITC-conjugated Donkey anti-Mouse Secondary Antibody (dilution, 1:200; Invitrogen) for 2 h at room temperature. DAPI (2 μg/ml, Research Organics, Cleveland, OH, United States) was used to counterstain the cell nucleus, and images were captured using CLSM.