Immunohistochemical localization of Nodal in endometrium across the menstrual cycle. (A) Late secretory human endometrial tissue (lanes 2 and 3) was analysed by reducing SDS-PAGE and Western blot using a Nodal-specific antibody. A 13 kDa band representing mature Nodal monomer and a 40 kDa band representing Nodal precursor were detected in the absence of a blocking protein (lane 2). The inclusion of a blocking protein significantly reduced the intensity of the Nodal bands (lane 3). Recombinant Nodal (lane 1) was included as a positive control. The Nodal antibody was used to localize Nodal in early proliferative (B), late proliferative (C), early secretory (D & G), mid secretory (E) and menstrual phase (F) endometrium. Note the precipitous decrease in Nodal staining within the stromal compartment during the early/mid secretory phases. In menstrual tissue, Nodal expression coincided with the expression of its antagonist, Lefty (H). Negative controls for immunostaining by replacement of antisera with goat IgG at a matching concentration are shown in insets. Arrowheads (G) indicate specific Nodal staining in endothelial cells during the early secretory phase. Bar, 50 μm. Scale bar on (F) relates to panels (B-F & H), whereas scale bar on (G) relates to panel (G). Tissue sections were also scored according to Nodal staining intensity within endometrial stroma (I) and epithelial glands (J). Intensity of staining is shown as individual (filled circles) and mean scores (I & J).