Heat-induced antigen-retrieved tissue sections were subjected to Vectastain Elite ABC-HRP system (PK6101, Vector Labs) for immunohistochemistry according to the manufacturer’s instructions. Primary antibodies were as follows: anti-Ki67 (1/10000; ab15580, Abcam), anti-Laminin (1/750; L9393, Sigma-Aldrich), anti-Perforin (1/200; ab16074, Abcam), and anti-PIBF1 (1/500; PMID:1463410734 (link)). In the case of staining Perforin, peroxidase-conjugated donkey anti-rat IgG (712-035-150, Jackson ImmunoResearch Laboratories) was applied as a secondary antibody. Labyrinthine Ki67-positive trophoblast cells or decidual Perforin-positive cells were counted in three randomly selected ×40 fields of each sample.
For immunofluorescence staining, antigen-retrieved tissue sections were subjected to primary antibodies, subsequently captured by Alexa Fluor-conjugated secondary antibodies. In vitro samples, including fixed cells and hHO cryosections, were permeabilized with 0.1% Triton X-100 and subjected to antibodies. The sections were mounted with DAPI (D1306, Molecular Probes), and examined under Zeiss LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany). CD31, cTnT, or PDGFRβ-stained areas were quantified using ImageJ software (NIH). The antibodies used for immunofluorescence staining are listed in Supplementary Data6 .
For immunofluorescence staining, antigen-retrieved tissue sections were subjected to primary antibodies, subsequently captured by Alexa Fluor-conjugated secondary antibodies. In vitro samples, including fixed cells and hHO cryosections, were permeabilized with 0.1% Triton X-100 and subjected to antibodies. The sections were mounted with DAPI (D1306, Molecular Probes), and examined under Zeiss LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany). CD31, cTnT, or PDGFRβ-stained areas were quantified using ImageJ software (NIH). The antibodies used for immunofluorescence staining are listed in Supplementary Data