Bouin s fixative

For quantification of metastatic lesions, mouse lungs were isolated, washed briefly in PBS, fixed in Bouin’s fixative (Sigma) overnight at 4 °C, and stored in 70% ethanol before counting lung nodules. For histological analysis, lungs were fixed in 10% formalin overnight at 4 °C, dehydrated, and embedded in paraffin (Tissue Tek Embedding Station). 3μm sections were cut on a Leica RM2255 rotary microtome. For antigen retrieval, deparaffinized slides were immersed in R-buffer A (Electron Microscopy Services) and boiled in a microwave for 20 min. Samples were blocked and permeabilized for 30 min in blocking buffer (5% normal goat serum, 0.3% TritonX-100 in PBS), followed by primary antibody incubation overnight at 4 °C using anti-CDH1 (1:100, BD Biosciences, 610181). Secondary antibody (1:500, BD Biosciences) was incubated for 1 h at RT. All sections were analyzed using a Nikon A1 confocal microscope and Zeiss fluorescence microscope.

Testes and cauda epididymides were fixed in Bouin’s fixative (41506; Sigma, Waltham, MA, USA) for 24 h. Next, the tissues were dehydrated in graded ethanol and embedded in paraffin. Five-micrometer sections were cut and mounted on glass slides, then stained with hematoxylin and eosin (H&E) or periodic acid schiff (PAS). Finally, images were obtained using a Nikon Eclipse TS100 inverted microscope (Nikon, Tokyo, Japan).

Haematoxylin and eosin staining was performed on 7 μm-thick cryosections fixed with 4% paraformaldehyde for 10 min at 4 °C. The staining was performed according to standard protocols. For Milligan’s trichrome staining, sections were fixed for 1 h with Bouin’s fixative (Sigma-Aldrich) and rinsed for 1 h under running water. Sections were then rapidly dehydrated to 95% EtOH in graded ethanol solutions, successively passed in 3% potassium dichromate (Sigma-Aldrich) for 5 min, rapidly washed in distilled water, stained with 0,1% acid fuchsin (Sigma-Aldrich) for 30 s, washed again in distilled water, passed in 1% phosphomolybdic acid (Sigma-Aldrich) for 3 min, stained with Orange G (2% in 1% phosphomolybdic acid) (Sigma-Aldrich) for 5 min, rinsed in distilled water, passed in 1% acetic acid (VWR) for 2 min, stained with 1% Fast Green for 5 min (Sigma-Aldrich), passed in 1% acetic acid for 3 min, rapidly dehydrated to 100% EtOH and passed in Xylene before mounting with Eukitt (Bio-Optica).

Diaphragm skeletal muscle was dissected from euthanized mice and fixed overnight at 4 °C in 4% paraformaldehyde and embedded in paraffin. For Mason’s trichrome staining, 5 μm sections were dewaxed in xylenes and rehydrated through graded alcohols and then post-fixed in Bouin’s fixative (Sigma-Aldrich, St. Louis, MO, USA). Sections were stained in modified Wiegert’s iron hematoxylin (Sigma-Aldrich), and then color differentiation was performed in acid alcohol followed by a trichrome stain. Sections were dehydrated through graded alcohols to xylenes, mounted, and immunohistochemistry (IHC) was performed. Sections were stained for CD3 (Abcam, 5690; 1:100, Cambridge, MA, USA), iNOS, Abcam 15,323; 1:100), and arginase-1 (Arg1) (Cell Signaling, 93,668, 1:200, Danvers, MA, USA). HRP-conjugated goat anti-rabbit IgG secondary antibodies were applied at 1:500 for 1 h. Only the HRP-conjugated secondary antibody, without a primary antibody, was used as a negative control for each stain. Antigens were revealed with ImmPACT DAB (Vector Labs, San Francisco, CA, USA), counterstained with Gil’s formula #3 Hematoxylin and mounted with Cytoseal XY. Random fields were imaged on each section using CellSens software and an Olympus BX50 microscope. The data are expressed as the average number of positive cells per 40× field. All counts were performed by blinded evaluators.

The mice were placed under a heat lamp or heat pad to dilate their tail veins. A temperature probe was placed in the box to ensure that the mice did not overheat. After approximately 10 min, each mouse was placed in a restraint to facilitate the intravenous injection of 2 × 10⁵ B16‐F10 melanoma cells suspended in 100 μL of 1× Hank's balanced salt solution. Injections were performed using 29½ G insulin needles (Terumo, Shibuya City, Tokyo, Japan). Fifteen days after the melanoma cell injection, the mice were killed and lungs with visible tumor nodules were collected, washed with 1× phosphate‐buffered saline and fixed in Bouin's Fixative (Sigma‐Aldrich, St. Louis, MO, USA). The tumor nodules in each lung were counted under a dissection microscope.

For histological analysis, testes and epididymides were collected and fixed in Bouin’s fixative (Sigma Aldrich) for 3 h at room temperature. After several washes in 70 % ethanol to remove excess stain, testes were embedded in paraffin. Tissues were sectioned at 5 mm thickness and stained by Periodic Acid-Schiff (PAS)-hematoxylin.