Alexafluor488 conjugated green goat antimouse igg

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).