B8895

Immunohistochemical analysis was performed on 10 μm fresh-frozen sections of P19 placental tissue blocks (n = 3 animals). Frozen sections were fixed with 4% paraformaldehyde/PBS, and endogenous peroxidase was quenched by immersing in 0.3% (v/v) hydrogen peroxide/methanol. A streptavidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) was used to block endogenous biotin according to the manufacturer's instructions. After 30 min of incubation with 10% normal goat serum, the sections were incubated at 4°C overnight with a rabbit anti-mouse CD11b polyclonal antibody (1:100 dilution, 0.5 mg/ml, ab75476, Abcam), a rabbit anti-mouse trophoblast specific protein alpha (TPBPA) antibody (1:100 dilution, 1 mg/ml, ab104401, Abcam), or a normal rabbit IgG (1:40 dilution, 0.4 mg/ml, sc-2027, Santa Cruz Biotechnology, Inc., Dallas, TX) as a negative control. Subsequently, the sections were incubated at room temperature for 1 h with a goat anti-rabbit IgG biotin conjugate (1:800 dilution, B8895, Sigma–Aldrich, St. Louis, MO). The immunoreactivity was visualized using the avidin-peroxidase (1:400 dilution, E2886, Sigma-Aldrich) and AEC substrate kit (Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer's instructions and then examined under light microscope (BX-51, Olympus, Tokyo, Japan).

Immunohistochemical staining of FFPE slides was performed by ARUP Laboratories using an automated immunostainer. Briefly, 5 μm tissue sections were obtained using a standard microtome and de-paraffinized with the EZ Prep solution. The slides were then treated with Cell Conditioning 1 (CC1), pH 8.5 for 68 min at 95 °C. The primary antibody (#ab80922, Abcam) was applied for 1 h at a dilution of 1:100 at 35 °C. Following removal of the primary antibody, the secondary antibody (#B8895, Sigma-Aldrich) was applied for 1 h at a dilution of 1:100 at 37 °C. Slides were then exposed to the IView DAB Map detection kit (Ventana) and counterstained with hematoxylin for 8 min. After dehydration in graded alcohol, coverslips were put on and slides were read by board-certified pathologists. Hematoxylin and eosin (H&E) staining was performed on 5 μm tissue sections.

PCNA is commonly used as a marker of cell proliferation in immunohistochemical studies due to its crucial role in DNA replication and repair [32 (link),33 (link)]. PCNA immunohistochemistry was performed according to our previously reported protocols [19 (link),20 (link),21 (link)]. Briefly, histological sections from six different animals per experimental group were investigated. Sections were incubated with rabbit anti-mouse PCNA monoclonal antibody (1:1000; D3H8P, XP Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA) in 1% BSA overnight at 4 °C. The sections were then treated with biotinylated goat anti-rabbit IgG antibody (1:900; B8895, Sigma-Aldrich, St Louis, MO, USA) followed by incubating with streptavidin-peroxidise conjugate (VectorLabs, Burlingame, CA, USA). The specific antigens were visualized with a diaminobenzidine (DAB) reaction (CellMarque, Sierra College Blvd Rocklin, CA, USA), counterstained with hematoxylin, and cover-slipped for microscopic examination. Negative controls were tested without the primary antibody. Proliferation was determined by counting the number of positively stained cells (brown nuclear reactivity) at 400× under standard light microscope. The mean percentage of proliferating cells was calculated from 4 to 6 representative fields per slide for each experimental group (n = 6 animals per group).