Openlab 3.1.7

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.

Images were acquired using HCImage software (Hamamatsu) and an Olympus SZX2 stereo microscope attached to a Lumen Dynamics X-Cite Fluorescence Illumination System for fluorescent detection assays. Animals were immobilized by chilling on NG agar plates to 4° prior to photographing. After initial image capture, subsequent analysis of images was performed using ImageJ software (NIH). For display of the injected animals, two consecutive images were overlaid using ImageJ. The scale bar was measured using a hemocytometer. For Figure 6, bright field and fluorescent images were acquired using Openlab 3.1.7 (Improvision), a C4742-95 CCD (Hamamatsu), and a Zeiss Axiophot.