E10.5-stage embryos were embedded in 10% gelatin, sectioned at 70 μm with a vibratome, and mounted with Fluoromount G (Southern Biotech, Birmingham, AL, USA). Older embryos and mammary glands were processed for X-Gal staining and fixation as described above. Isopropanol was substituted for xylene to prevent diffusion of the X-Gal stain during processing and tissues were embedded in paraffin and sectioned. Sections (4 μm) were placed on Superfrost Plus slides, baked 1 h at 60°C and deparaffinized for 5 minutes in Citrisolv for X-Gal-stained tissues. Tissues were then rehydrated through a reverse gradient of ethanol solutions. For histology, sections were stained with 0.1% solution of Nuclear Fast Red (NFR) (Polyscientific, Bayshore, NY, USA) for 1 minute. Tissues were then dehydrated and dipped in xylene (or Citrisolv in the case of X-Gal-stained tissues) before being mounted in Cytoseal (VWR). For immunohistochemistry (IHC), citric acid antigen retrieval was performed by submerging the slide containing deparaffinized 4-μm sections in 10 mM sodium citrate solution (pH 6.0) and boiling in a microwave at 90% power for 30 minutes, followed by quenching of endogenous peroxidase using 3% hydrogen peroxide. Primary mouse antibodies against smooth-muscle actin (SMA) 1 (1:500, DAKO, Carpinteria, CA, USA), estrogen receptor (1:500, DAKO), p63 (1:1,000 LabVision, Kalamazoo, MI, USA), and rabbit antibodies against Cytokeratin 14 (1:8,000, Covance, Princeton, NJ, USA), Lef-1 (1:100 Cell Signaling, Danvers, MA, USA), androgen receptor (1:500, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and guinea pig antibodies against Vimentin (1:1,000, Progen) were added overnight at 4°C. For IHC, biotin-labeled secondary antibodies (1:1,000) and streptavidin-horseradish peroxidase (HRP) (1:200, Vector Laboratories, Burlingame, CA, USA) were added for 30 minutes each, and colorimetrically detected with diaminobenzidine (Vector Labs). Frozen 5-μm sections were stained with rabbit antibodies against LTBP (Ab39 [
43], 1:200, a gift from Dr Carl-Henrik Heldin, Uppsala University, Sweden, and rL1C [
44], 1:100, a gift from Dr Lynn Sakai, Portland Shriners Research Center, Portland, OR, USA), tropoelastin (1:500, Elastin Products Company, Inc., Owensville, MO, USA), and mouse anti-SMA, described above, were detected by Cy3-labeled donkey anti-rabbit (Fisher Scientific) and Alexafluor-488-labeled donkey anti-mouse secondary antibodies (Life Technologies Inc, Carlsbad, CA, USA). Bioreagent (4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) from Sigma Aldrich) was used for immunofluorescent localization of nuclei in confocal images. Elastic fibers were also detected by staining with Wiegert’s resorcin-fuchsin for 1 minute [
45].