Immunohistochemistry and in situ hybridization to detect human albumin expression in human cells.
(A-B) Human albumin expression in the livers of chimeric NOD/SCID/β2M-null mice that had received liver injury with or without rhHGF. (A) Slides were made from paraffinized liver sections prepared from the chimeric and control mice, and immunohistochemistry was performed using an antihuman albumin antibody that did not cross-react with the murine hepatocytes (A). The antibody was visualized with a blue chromogen. Panel A shows a low magnification image of a control slide prepared using the same reagents. Panels B-C show low and high magnifications of the albumin-expressing human cells that were detected distributed throughout certain regions of the chimeric liver from one of the mice that had received recombinant human HGF after CCl4-mediated liver injury. Specific areas of the chimeric livers appeared heavily repopulated, as shown, although other areas were nearly devoid of human albumin–expressing cells. Panel B: (i-ii) High magnification of human albumin–expressing cells identified with an antihuman albumin-FITC antibody in liver sections. Nonalbumin-expressing human or mouse cells are seen surrounding the albumin–expressing cells. The background level of autofluorescence in injured tissues can be seen in the control sections (iii-iv) taken from mice that had received both CCl4 and HGF but no human stem cell transplantation. (C) Human cord blood stem cell–derived albumin-expressing cells recovered from the livers of injured, chimeric NOD/SCID/β2M-null mice. To reduce the chance of artifacts appearing from overlaid cells, cytospin slides of dissociated hepatocytes were prepared. Slides were stained with antihuman albumin-FITC Ab, and then in situ hybridization was performed using a probe specific for human DNA Alu sequences. The upper panel shows a brightfield image (left) and the fluorescent image (right) with an overlay image in the center. Human hepatocyte–like cells appear green in the cytoplasm, detected by the antihuman albumin-FITC antibody, and black in the nuclei, as detected by DAB reaction for human Alu sequences. Human blood cells showed black nuclei only (Alu+) without cytoplasmic albumin expression. Original magnifications: × 10 (A); × 100 (B); and × 20 (C).
