Mz16f stereoscope

The fourth mammary glands were harvested from virgin to involution stages of miR-205^fl/fl mice and then fixed in 2% paraformaldehyde for 3 hours at 4°C before staining in X-Gal staining solution (containing 1 M MgCl₂, 5 M NaCl, 1 M pH 7.9 HEPES, 30 mM K₄Fe₂(CN)₆•3H₂O, 30 mM K₃Fe₂(CN)₆, 10% NP-40, 1% X-Gal solution in DMF). After the 1X phosphate buffered solution wash that followed the X-Gal staining step, tissues were sequentially dehydrated, fixed (Carnoy’s fixative), and rehydrated for nuclear fast red staining. Upon detection of the red counterstain, tissues were dehydrated and cleared in Histo-Clear overnight. Whole-mount images were acquired by spreading the tissues out between two glass slides and imaging on a Leica MZ16F stereoscope.

Kidneys were dissected from E11.5 embryos and ureteric buds were isolated from the surrounding mesenchyme as described previously (Costantini et al., 2011 ). The isolated ureteric buds were cultured in matrigel supplemented with D-MEM/F12 (Gibco) containing 10% FBS (Gibco), 200 nM cis and trans retinoic acid, and 100 ng/mL recombinant rat GDNF (as recommended by Prof. Frank Costantini’s lab; personal communication). Images were taken at 24 h time intervals with a DFC300 FX camera attached to a Leica MZ16F Stereoscope.

All steps were performed at room temperature. Embryos were fixed in 95 % ethanol overnight. Samples were then placed in acetone overnight. Embryos were then placed in alcian blue (Sigma A5268) dissolved in 80 % ethanol, 20 % (glacial) acetic acid at a concentration of 0.03 % overnight. The embryos were then de-stained by two thirty minute washes in 70 % ethanol and then incubated them in 95 % ethanol overnight. The embryos were pre-cleared in a 1 % potassium hydroxide (KOH) (Fisher Scientific 1310–58-3) solution for one hour. Embryos were then placed in a 0.005 % alizarin red (Sigma A5533) dissolved in 1% KOH overnight. The embryos were then placed in a 50 % glycerol (Fisher 56-81-5): 50 % (1 %) KOH solution until clear. The average time was one week to become fully cleared. Once cleared, the embryos were then placed in 100% glycerol for long-term storage and imaging. Skeletal preparations were imaged Leica MZ16F stereoscope and Leica DFC490 camera with Leica software.

The fourth pair of mammary glands were harvested from mice and spread onto glass slides. Next, they were fixed in Carnoy’s Fix (6:3:1 mixture of 100% ethanol, chloroform, and glacial acetic acid) for 1–4 hr, and stained in carmine-alum stain overnight. The stained glands were washed and dehydrated in 70%, 95%, and 100% ethanol for 1 hr each, and then transferred to xylene overnight for clearing. They were mounted in Poly-Mount Xylene (Polysciences, Inc) and imaged using a Leica MZ16F Stereoscope with a Leica DFC300FX camera.