N histofine

FFPE tissues were sectioned at five-micron thickness and labeled with IL-4Rα (MAB230, R&D Systems), IL-13Rα1 (AF152, R&D Systems), or IL-13Rα2 (AF146, R&D Systems) antibodies (1:100) using AEC solution and N-Histofine (universal immune-peroxidase polymer, anti-goat/anti-mouse, Nichirei Biosciences INC). IL-4Rα, IL-13Rα1, and IL-13Rα2 expression was detected using the universal immune-peroxidase polymer kit according to manufacturer’s protocol. Appropriate negative (no primary antibody) tissues were stained in parallel. CD8 expression levels were assessed on whole tissue sections using ImmunoMembrane [53 (link)], an ImageJ plugin that uses color deconvolution for stain separation and a customized algorithm for cell membrane segmentation. A quantitative score (IM-score, 0–20 points) is generated according to the membrane staining intensity and completeness. Specimens are classified into 0/1+, 2+ or 3+ based on IM-score cut-offs. The classification and membrane segmentation are presented as a pseudo-colored overlay image (S1 Fig). ImmunoMembrane is freely accessible at: http://jvsmicroscope.uta.fi/immunomembrane/.

Bovine mammary samples were fixed in Bouin’s solution, dehydrated in a graded ethanol series (50% to 100%), cleared in xylene and embedded in paraffin. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E; Sigma) to visualize the morphology of the mammary layers.
Immunohistochemistry was performed on the 5-μm paraffin-embedded sections after antigen retrieval. The reaction with primary antibodies was initiated by blocking the sections with 10% bovine serum albumen and 5% goat serum in 0.1% PBST for 1 h prior to overnight incubation with the first antibody at 4°C. Sections were washed and incubated with N-Histofine (Nichirei Biosciences, Tokyo, Japan) for 30 min at room temperature. Signals were generated with 3,3-diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA). For immunofluorescence analyses, the sections were incubated with secondary antibodies for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Qbiogen, Irvine, CA). The antibodies and their dilutions are listed in S1 Table. In all of the analyses, stained tissues were visualized and photographed under an inverted fluorescence microscope (Eclipse Ti, Nikon Instruments, Melville, NY) equipped with NIS-Elements AR 3.2 imaging software (Nikon Instruments), or an Olympus IX 81 inverted laser scanning confocal microscope (FluoView 500, Tokyo, Japan).