Phospho h3 ser10

Term placenta samples were collected through the Preeclampsia And Non-preeclampsia Database (PANDA^{21 (link)}) an obstetrical biosample effort approved by the medical ethical committee of the Academic Medical Center. First trimester placenta samples were collected as described above. First and third trimester placenta tissue and first trimester placental explants were sectioned and subjected to standard immunohistochemistry procedures. Antigen retrieval was performed using microwave pre-treatment in sodium citrate buffer (antigen retrieval was omitted when using the ELABELA antibody). Blocking was done in 5% BSA, followed by overnight primary antibody incubations in 1% BSA at 4 °C. The following primary antibodies were used: ELABELA (custom by^{11 (link)}), APLNR (Sigma, SAB2700205), Apelin (GeneTex, GTX37465), HLA-G (Novus Biologicals, NB500-302) and phosphoH3(Ser10) (Sigma, 09-797). Finally, Powervision Poly-HRP secondary antibodies were used followed by DAB staining and counterstaining using haematoxilin. ImageJ was used to count the number of proliferating extravillous trophoblasts. For immunofluorescence, HTR8/SVneo cells were fixed, followed by blocking in 3% BSA. Primary antibodies were added overnight at 4 °C. Finally, Alexafluor-488 labeled secondary antibodies were used followed by DAPI counterstain.