*4.4. Immunofluorescence and Immunohistochemistry*

Implantation sites with myometrium attached for immunofluorescence (IF) and immunohistochemistry (IHC) were fixed in 4% paraformaldehyde at 4 ◦C, infiltrated with 30% sucrose in PBS, embedded in Tissue-Tek®O.C.T™ Compound (Sakura Fine Technical Co. Ltd., VWR, Radnor, PA, USA), and cryosectioned at 7 µm. Interembryonic regions and central parts of the decidua were confirmed by H&E staining of every 5th section. For each implantation site, immunostaining was performed, as previously described [27,45] on at least 3 sections that were equally spaced along central parts of the decidua. The specificity of Notch protein and ligand primary antibodies was previously determined [27,58]. Slides stained with secondary antibody alone served as negative controls for IF and IHC staining. Antibodies are listed in the Supplementary Table S1. For colorimetric IHC, biotinylated anti-goat (Vector BA-5000, 1:400) or biotinylated anti-rat (Vector BA-4001, 1:200), the avidin/biotin blocking kit (Vector SP-2001), the Vectastain ABC kit and DAB substrate kit (Vector SK-4100) were used. For IF, Vectashield containing 40, 6-diamidino-2-phenylindole (DAPI) (Vector) was used for nuclear visualization and mounting. For each experiment, 2–3 implantation sites per pregnant dam were analyzed.
