Anti mouse igg conjugated to hrp

Placentation sites were embedded in OCT compound and sectioned at 10 μm thickness. Sections were fixed in 4% paraformaldehyde, washed in phosphate-buffered saline (pH 7.4) three times for 5 minutes each, blocked with 10% Normal Goat Serum (cat. no. 50062Z, Thermo Fisher), and incubated overnight with a mouse monoclonal anti-pan cytokeratin antibody conjugated with fluorescein isothiocyanate (1:300 dilution; cat. no. F3418, Sigma-Aldrich) to identify trophoblast cells, or with a mouse monoclonal anti-vimentin antibody (1:300 dilution; cat. no. sc-6260, Santa Cruz Biotechnology) to distinguish placental compartments followed by incubation with anti-mouse IgG conjugated to HRP (1:500 dilution, cat. no. A9044, Sigma-Aldrich) for 3 hours at room temperature and color development with an AEC substrate kit (cat. no. SK4200, Vector Laboratories). Sections were then mounted with Fluoromount-G mounting media (cat. no. 0100–01, SouthernBiotech, Birmingham, AL) and examined microscopically. Fluorescence images were captured on a Nikon 80i upright microscope (Nikon) with a Photometrics CoolSNAP-ES monochrome camera (Roper). The area occupied by cytokeratin-positive cells (invasive trophoblast cells) within the metrial gland was quantified using ImageJ software, as previously described.⁵⁷