*4.4. RNA-Scope*

RNAscope was performed by the Comparative Pathology Core at Iowa State University College of Veterinary Medicine using the RNAscope 2.5 High Definition (HD)-Red Assay (Advanced Cell Diagnostics Cat #322350) according to manufacturer's instructions. Briefly, for each biological replicate, e9.5 implantation sites were dissected from timed-pregnant CD-1 mice obtained from Charles Rivers Labs. Each implantation site was embedded in paraffin and was cut at 5 µm thickness, mounted, deparaffinized and treated with hydrogen peroxide prior to target retrieval, and then treated with protease plus. Mm-Plagl1 (cat # 462941) and control probes were applied to slides for hybridization followed by six rounds of amplification. The signal was detected with Fast RED and slides were counterstained with hematoxylin. *Plagl1* signal appears as red on the slide. Control slides included positive control probe Pentidylpropyl isomerase B (Mm-PPIB cat # 313911) used to determine RNA viability in the samples, and negative control probe DapB (cat # 310043) to detect nonspecific staining.
