Evos fl auto 2 microscope

Carmine alum whole mounts were carried out as previously described [16 (link), 17 (link)]. Briefly, fourth inguinal mammary glands were fixed in 10% neutral‐buffered formalin (NBF) (Leica) overnight at 4°C. Glands were dehydrated for 1 h in distilled water, then 1 h 70% ethanol and 1 h 100% ethanol before incubation in xylene overnight (VWR international). Rehydration was achieved by 1‐h incubation in 100% ethanol, 70% ethanol and distilled water, before staining with Carmine Alum solution at room temperature overnight (0·2% (w/v) carmine and 10 mM aluminium potassium sulphate (Sigma)). Tissue was dehydrated again and incubated overnight in xylene. Glands were then mounted with DPX (Leica), and 10× magnification stitched brightfield images were obtained using an EVOS FL auto2 microscope (Thermo Fisher). 5× brightfield images were obtained using the Zeiss Axio Imager M2 with Zen 2012 software. TEBs were counted as the average from at least 2 F.O.V. from each whole mount. All samples were blinded before measurements were taken.

Carmine alum wholemounts were prepared as described previously (Wilson et al., 2017 (link)). Briefly, fourth inguinal mammary glands were spread onto Superfrost Plus slides (VWR) and fixed overnight in 10% neutral buffered formalin (NBF) (Leica) at 4°C. Glands were dehydrated for 1 h in distilled water, followed by 70% ethanol and 100% ethanol before overnight incubation in xylene (VWR international). Tissue was rehydrated by a 1 h incubation in 100% ethanol, 70% ethanol and distilled water, before staining in carmine alum solution overnight at room temperature [0.2% (w/v) carmine and 10 mM aluminium potassium sulphate (Sigma)]. Tissue was dehydrated again before overnight incubation in xylene. Finally, glands were mounted with DPX (Leica) and stitched bright-field images at 10× magnification were taken using an EVOS FL auto2 microscope (ThermoFisher). Ductal elongation, and branched area from the lymph node, were measured using ImageJ 1.52a (Schneider et al., 2012 (link)). Bright-field images at 5× magnification were obtained using the Zeiss Axioimager M2 with Zen 2012 software. The numbers of branches and branch thickness were counted as the average from three measurements from six individual fields of view (FOV) from each wholemount. TEBs were counted as the average from at least two FOV from each wholemount. Sample identities were hidden before measurements were taken.

Thirty thousand PAAFs were plated onto 35 mm cell culture dishes with #0 coverglass bottom (#D35-20-0-N, CellVis, Sunnyvale, CA, USA) onto 0.5 kPa or 3 kPa polyacrylamide gels, or directly onto plastic for 3 days at 37 °C and 5% CO₂. Images were taken on an EVOS FL Auto 2 microscope, running software v2.0.1732.0 (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies against Smooth Muscle alpha-Actin (mouse #A5228 1:100, Sigma, St. Louis, MO, USA) with secondary Goat anti-Mouse Texas Red (#T862, 1:250, Life Technologies, Carlsbad, CA, USA), Wheat Germ Agglutinin-488 for membrane (#W6748, 10 g/mL, Life Technologies, Carlsbad, CA, USA) and DAPI for nuclei in mounting media with Prolong Gold Antifade Reagent with DAPI (#P36941, Life Technologies, Carlsbad, CA, USA). Images were processed using DeconvolutionLab2 (EPFL, Lausanne, Switzerland) in ImageJ v1.53g4 developed by the National Institutes of Health (Bethesda, MD, USA).

Three sections per biopsy were tiled at 20× magnification using an EVOS FL Auto 2 microscope (ThermoFischer Scientific). All new images were captured as 24‐bit RGB color images with 3.2 million pixels (12 MB) at a resolution of 58,522 pixels per inch. Following image collection, images were manually parsed into “edge” versus “continuous” images to distinguish images that were wholly contained within the section (continuous) versus images that were partially tissue and partially blank space (edge). Edge images were excluded to avoid artifacts in analysis. Continuous images were then loaded into the program for quantification.

H&E staining of mouse organs and tumours was performed by the staff at the Anatomical Pathology Laboratory Services located at The Royal Children’s Hospital, Melbourne, Australia according to the standard H&E protocol as described previously [28 (link)]. Paraffin embedded tissues were sectioned at 4 µm thickness, deparaffinised, rehydrated and stained for 3 min with Haematoxylin (Australian Biostain Pty Ltd, Traralgon, VIC, Australia). Slides were rinsed with 0.25% Acid Alcohol and Scott’s Tap water Substitute. Sections were stained with Eosin (Amber Scientific, Midvale, WA, Australia) for 2 min before final rinsing with absolute alcohol and xylene. The metastatic development was assessed by a qualified pathologist at The Royal Women’s Hospital (Melbourne, VIC, Australia). Histological images of stained organs and tumours were taken using Evos FL Auto 2 microscope (Thermo Fisher Scientific).