Diaminobezidine

The expression of the trophoblast marker, cytokeratin 7 (CK7) [18 (link)], the proliferative maker, Ki67, and the EVT makers, human leukocyte antigen (HLA)-G [19 (link)] and integrin α5 [18 (link)] in the chorionic villous explants was examined by IHC at 72 h after lentiviral transduction. The explants were fixed, paraffin embedded and sectioned. Following antigen retrieval, the sections were incubated sequentially with 3% H₂O₂, goat serum, and a specific primary antibody overnight at 4°C including anti-CK7 (1:100; Bioss, Beijing, China), anti-Ki67 (1:100; Bioss), anti-HLA-G (1:50; Santa Cruz) and anti-integrin α5 (1:200; Boster, Wuhan, China). Thereafter, the sections were incubated with biotin-conjugated goat anti-mouse/rabbit IgG and HRP-labeled streptavidin (Beyotime). Diaminobezidine (Sigma-Aldrich) was added for the chromogenic reaction, and the cell nuclei were stained with hematoxylin. The sections were mounted and observed under an Olympus DP73 microscope at 400× magnification.

Histologic alteration of tight junction proteins was examined using immunohistochemistry. Samples of the kidney was embedded in paraffin, cut into 5- μm sections, deparaffinized with xylene, and hydrated in descending graded ethanol solutions. The sections were then mounted onto glass slides (Matunami, Ishikawa, Japan). Endogenous peroxidase activity was blocked by 3% hydrogen peroxidase in PBS for 30 minutes at room temperature. To prevent non-specific reactions, the sections were incubated with 10% goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS for 1 hour at room temperature. After washing with TBS-T, the sections were incubated O/N at room temperature with the same primary antibodies used for Western blotting (rabbit anti-CLDN4 and - CLDN16; and mouse anti-CLDN1) diluted 1:250 with 5% BSA. The slides were washed with TBS-T before being incubated with biotinylated secondary antibodies (1:500, rabbit or mouse IgG; Vector Laboratories, Inc.) for 1 hour at 37°C and then ABC Elite solution (Vector Laboratories, Inc.) for 30 min at 37°C. Diaminobezidine (Sigma, St. Louis, MO, USA) was used as a chromogen. The sections were counterstained with hematoxylin and mounted in Cytoseal*60 (Richard-Allan Scientific Co., Kalamazoo, MI, USA).