Fig. 4. Block in secretory activation at the transition to lactation. (A,B) Hematoxylin and Eosin stained sections at day 18 of gestation. Scale bar: 50 µm. Note the uniform size of alveoli and the extent of differentiation in the wild-type glands (A), versus the mixture of collapsed, non-differentiated and differentiated alveoli in the Hif1a^–/– glands (B). (C) Expression of Cre (brown, nuclear staining) detected in the Hif1a^–/– gland shown at higher magnification in D. Areas that expressed Cre contained small, relatively undifferentiated alveoli (null; white arrows), whereas areas negative for Cre achieved differentiation (WT; black arrows). (E-G) Multiple antigen labeling immunostaining was performed to sequentially detect Glut1 (brown stain) and Cre (purple stain, nuclear localization) using paraffin wax-embedded sections at day 18 of gestation followed by counterstaining with Methyl Green. The black arrows indicate the robust, uniform Glut1 staining pattern observed in wild-type, differentiated alveoli. The white arrows note the relatively weak Glut1 expression detected in alveoli that also express Cre (Hif1a^–/–). Wild-type (F) and Hif1a null alveoli (G) were individually imaged at higher power to highlight the reduced expression of Glut1 in response to deletion of Hif1a. (H) Average Chalkley score following anti-CD31 immunostaining to determine MVD at day 18 of gestation, (mean±s.e.m.). (I) DNA, RNA, and protein content per gram tissue at day 18 of gestation (mean±s.e.m.).

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