Oro stock solution

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.

Cultured cells were washed with phosphate-buffered saline and fixed in 10% neutral-buffered formalin at room temperature for 1 h. The cells were then stained with 0.5% filtered ORO stock solution (Sigma, Saint Louis , MO, USA) in isopropanol. To evaluate the intracellular triglyceride content, the stained cells were redissolved with isopropanol. The absorbance at a wavelength of 520 nm was measured.