Endometrial NSD2 and H3K36me2 in Recurrent Implantation Failure

Proliferative-phase endometrial tissues from 10 patients with RIF and 10 FER controls were collected and washed with PBS immediately. Tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and serially sectioned. Immunostaining was performed according to the instruction of the mouse and rabbit specific HRP/diaminobenzidine (DAB) (ABC) Detection IHC Kit (ab64264, Abcam). The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C. The primary antibodies used in the study were all commercial antibodies reported by other empirical studies (Zhu et al. 2019 (link), Dai et al. 2020 (link), Ganig et al. 2021 (link), Acke et al. 2022 (link)). For each immunohistochemical run, a slice from each group was randomly selected as the negative control. The negative control was tested by incubation with immunoglobulin G (IgG) from the corresponding species. Next day, they were incubated with secondary antibodies, stained with DAB, and counterstained with hematoxylin. The slides were then dehydrated, cleared, and mounted. The images were captured under a microscope. See Supplementary methods for the immunohistochemical scoring method.