Goat serum blocking buffer

After 7-day post-transplantation to the injured NOD/SCID mice liver, donor hUCMSCs proliferation was quantified by immunohistochemical staining of PCNA. Fresh liver tissues were embedded with OCT medium and “snap-frozen” in dry ice. Frozen sections of 10-μm thickness were prepared and subjected to permeabilization in acetone at –20 °C for 10 min. To reduce non-specific signal, slides were incubated with goat serum blocking buffer (Boster, Wuhan, China) at room temperature for 1-hour. Subsequently, the slides were incubated with primary antibodies PCNA (1:100, Cell Signaling). After washing thrice with PBS, slides were incubated with mouse antibody against mouse IgG conjugated with Alexa flour (1:1000, Cell Signaling). Sections were co-stained with human cytokeratin-18 (hCK-18; 1:100, Abcam HK, NT, HK) and goat antibody against rabbit IgG conjugated with FITC (1:1000, Abcam HK). Apoptosis was quantified by terminal dUPT nick end-labeling (TUNEL) using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Billerica, MA) after 3-day post-transplantation. The number of PCNA cells or apoptotic cells was quantified in 3 microscopic fields at ×40 magnification using ImageJ software.

Liver tissue samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and processed for immunological and histological assays. Five-micrometer tissue sections were cut and stained with hematoxylin and eosin (H&E) or antibodies of hCK-18 and hAFP. For the evaluation of key proteins in host hepatocytes and transplanted stem cells, fresh liver tissues were embedded with optimal cutting temperature (OCT) medium and “snap-frozen” in dry ice. Frozen sections of 10-µm thickness were prepared and subjected to permeabilization in acetone at −20°C for 10 min. To reduce non-specific signal, slides were incubated with goat serum blocking buffer (Boster, Wuhan, China) at room temperature for 1-hour. Subsequently, the slides were incubated with primary antibodies PCNA (1∶100, Cell Signaling Technology, Danvers, MA) and hHGF (1∶500, Takara, Shiga, Japan), respectively. After washing thrice with PBS, slides were incubated with goat antibody against rabbit IgG conjugated with FITC (1:1000, Abcam HK) or mouse antibody against mouse IgG conjugated with Alexa flour (1:1000, Cell Signaling) at room temperature for 1-hour. Hoechst was applied to counter-stain the nuclei at room temperature for 15-minute before examination.