To determine the expression of specific markers, paraffin-embedded sections of mouse mammary glands or co-cultures were analyzed by immunohistochemistry. Briefly, sections were deparaffinized in xylene, hydrated in a series of graded alcohols (100%, 95%, 70% and 50%), and treated with antigen unmasking solutions (Vector Laboratories, Inc. Burlingame, CA, USA) or with Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0) heated to 95–100 °C in a pressure cooker. Sections were blocked with nonimmune goat or horse serum (depending on the host species (goat or horse, respectively) of secondary antibodies used for immunofluorescence staining), and processed for immunofluorescence staining as described below.

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