Cellsens software

Frozen sections were thawed, fixed in 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X100 in PBS for 10 minutes. Paraffin-embedded sections were deparaffinized in xylene washes, rehydrated in a ethanol gradient and washed in PBS. Slides were immersed in Antigen Unmasking Solution (Vector Labs) and steamed for 15 minutes. If blocking of endogenous peroxidase activity was required, slides were incubated in a 3% H₂O₂ solution for 15 minutes. Sections were blocked in 5% goat serum for 20 minutes and incubated with primary antibody overnight. Biotinylated secondary antibody was added the next morning, with anti-K14 if required. For immunofluorescence analysis, the appropriate streptavidin-fluorescent conjugate was added, slides were mounted with Vectashield mounting medium containing DAPI and sealed with nail varnish. Images were captured on a CKX41 microscope using cellSens software (Olympus). For immunohistochemistry, streptavidin-horse radish peroxidase (Vector Labs) was added and peroxidase was detected using diaminobenzidine (Vector Labs). Sections were counterstained with hematoxylin, dehydrated and mounted with xylene-substitute mounting medium (Shandon). Images were taken on a BX43 microscope using cellSens software (Olympus). All antibody/conjugate incubations were performed in 3% BSA in tris buffered saline with tween.

Primary mammary tumors were cut at the time of sacrifice, fixed in 10% formalin, transferred to 70% ethanol, embedded in paraffin, and sectioned at 5micron. The sections were deparaffinized, rehydrated and subjected to antigen retrieval as previously described (44 (link)). Primary antibodies anti-Vimentin (Cell Signaling, Danvers, MA) and secondary AlexaFluor568-conjugated (red) donkey antirabbit IgG (1:500, Molecular Probes, Eugene, OR) and AlexaFluor488-conjugated (green) goat antimouse IgG (1:500; Molecular Probes) for 2h were used. Nuclei were counterstained with 0.2μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich, St. Louis, MO). Sections were mounted using Fluorogel mounting medium (Electron Microscopy Sciences, Hatfield, PA). An Olympus AX70 fluorescence microscope (Olympus, Center Valley, PA) was used to capture images from immunofluorescence staining with CellSens software (Olympus, Center Valley, PA) software. At least six individual ×400 fields per group were captured for counting Vimentin-positive cells and the total number of DAPI-positive cells. Quantification of the intensity of the Vimentin-positive cells was performed using CellSens software (Olympus, Center Valley, PA).

Epididymal (eWAT) depot and interscapular brown adipose tissue (BAT) were fixed in 4% paraformaldehyde for 48 hr, embedded in paraffin, cut into 5-mm sections, and stained with Hematoxylin and Eosin (H&E). The total number of adipocytes was measured using the CellSens Software (Olympus). Adipocyte number per area was averaged from at least 500 adipocytes counted from 12–15 images per animal. Crown-like structures were counted on paraffin-embedded and H&E stained eWAT sections. Paraffin-embedded eWAT sections were stained with an anti-F4/80 antibody to detect macrophages in the adipose tissue. The images were acquired with an XM10 Olympus fluorescent camera (Olympus). F4/80 immunofluorescence staining was quantified based on color intensity using the CellSens Software (Olympus) in representative nonconsecutive fields per slide from three slides per animal in a blinded fashion.