*4.4. Immunohistochemistry*

For immunohistochemical (IHC) analysis, tissues were fixed in methyl Carnoy's solution and embedded in para ffin, as previously described [67]. Each group consisted of 6 placentas (3 sections of the central region, each) from 2 dams. Two-micrometer para ffin sections were prepared with a HM340E microtome (Thermo Fisher Scientific, Waltham, MA, USA). After de-para ffinization and rehydration with intermittent Tris-bu ffered saline (TBS) washing, tissue sections were unmasked by cooking in target retrieval solution (TRS, Dako Agilent, Santa Clara, CA, USA) for 10 min. Endogenous peroxidase activity was blocked with 3% H2O2 for 20 min at room temperature. Sections were then incubated in fetal calf serum (FCS) at 37 ◦C for 30 min and coated with the primary antibody (Rarres1: MyBioSource, San Diego, CA, USA, 1:50; Rarres2: Thermo Fisher, Waltham, MA, USA 1:100). After incubation at 4 ◦C overnight, sections were washed in TBS and layered with the secondary antibody (dilution 1:500; biotin-conjugated, goa<sup>t</sup> anti-rabbit immunoglobulin G; Vector Laboratories, Burlingame, CA, USA) at room temperature for 30 min. Subsequently, sections were incubated with avidin-biotinylated horseradish peroxidase complex (Vectastain PK-6100; Vector Laboratories) at RT for 30 min and with a DAB (diaminobenzidine tetrahydrochloride) kit (SK-4100; Vector Laboratories, both supplied by Linaris, Dossenheim, Germany) for 15 min and counterstained with hematoxylin (Merck, Darmstadt, Germany). After embedding in Entellan (Merck, Darmstadt, Germany), imaging was performed with a DMC 6200 camera mounted on a Leica DMR microscope (Type 020-525.731) using LASX 3.4.2.18368 image software (all from Leica Microsystems, Wetzlar, Germany). Representative photomicrographs for antibody specificity testing are shown in supplementary Figure S1.
