Mammary tissue was formalin fixed (4% v/v), paraffin embedded before sectioning (5 μm), and subjected to standard H&E staining. Histology was imaged with a Zeiss
Axioscop2 microscope using PlanNeoFluar 40× lens (numerical aperture 0.75) fitted with a Zeiss AxioCam color camera, and analyzed with
Openlab (3.1.7) software (Improvision).
Whole-mount analysis was performed by spreading inguinal mammary glands on polysine slides and stained with carmine alum as described previously (Akhtar et al., 2009 (
link)).
ORO staining was performed by staining 10 μm mammary cryosections in freshly diluted ORO solution (6 parts 0.5%
ORO stock solution [Sigma] and 4 parts water) for 15 min. Sections were rinsed twice with 60% isopropanol, once with water, and then counterstained with Mayer's hematoxylin for 1 min before photography as above.
Akhtar N., Li W., Mironov A, & Streuli C.H. (2016). Rac1 Controls Both the Secretory Function of the Mammary Gland and Its Remodeling for Successive Gestations. Developmental Cell, 38(5), 522-535.