Immunohistochemistry of Placental Tissue

Immunohistochemistry procedure was performed as previously described^{5 (link)}. Briefly, paraffin embedded human placental tissue samples were sectioned, deparaffinized, and hydrated with Histoclear and ethanol (Sigma, St. Louis, MO, USA). Slides then underwent antigen retrieval using 10 mM sodium citrate (pH 6) buffer in a pressure cooker. Sections were blocked using DAKO Dual Endogenous Enzyme Block for Autostainer and 2.5% normal horse serum (Agilent, Santa Clara, CA, USA). Slides were then incubated with primary antibody m28-RNLS (1:500) overnight at 4 °C and secondary antibody IMPRESS Reagent anti-Rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Antibody binding was detected using Vector DAB substrate kit and counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA). Images were obtained at 20× and 60× using Echo^® Revolve microscope. Staining specificity was determined by labeling with secondary antibody.