Cryomatrix

Fibroblasts were labeled using a PKH26 red fluorescent cell membrane labeling kit (Sigma,
St. Louis, MO, USA) according to manufacturer’s instructions and injected IP into mice.
Mice were euthanized at six time points at 7-day intervals (i.e. week 1 to week 6). Cells
were retrieved from peritoneal cavity (by lavage), lymph nodes, spleen, lung, and skin.
Single cell suspensions form tissues were prepared using collagenase D (Roche Diagnostics)
digestion (1 mg/ mL, for 30 min at 37 °C) and examined using flow cytometry. To further
characterize the cells that were extracted from lymph nodes, these cells were stained for
CD90.2 (eBioscience) and examined using flow cytometry. Presence of migratory fibroblasts
in lymph nodes was examined using confocal microscopy. To do so, PKH26-labeled
IDO-expressing fibroblasts (2 × 10⁷ cells/mouse) were injected IP into C3 H
mice. After 2 weeks, mice were euthanized and mesenteric lymph nodes were harvested,
freshly frozen and embedded in Cryomatrix (Thermo Scientific, Kalamazoo, MI, USA). Frozen
sections (5 μm thick) from lymph nodes were permeabilized with 0.1% Triton-X-100 in
phosphate buffered saline (PBS), stained with 4’,6-diamidino-2-phenylindole (DAPI) and
visualized using a confocal fluorescence microscope (Axio Observer Z1 inverted confocal
with spinning disk, Carl Zeiss, Jena, Germany). Images were analyzed using Zen software
(Carl Zeiss).

The mice were euthanized at different time points (n=6 animals per group at each time point). The corneas were fixed in 4% paraformaldehyde, transferred to 30% sucrose/PBS overnight at 4 °C, and embedded at optimal cutting temperature (OCT; Cryomatrix, Thermo Scientific, Montigny-Le-Bretonneux, France). Ten-micrometer frozen sections were washed with PBS, and blocked for 1 h with 0.1% Triton-1% BSA-PBS, before overnight incubation at 4 °C with rabbit anti-dystrophin H4 antibodies (1:2,000) [12 (link)]. The sections were then incubated with a goat anti-rabbit, goat anti-mouse, and goat anti-rat secondary antibody (1:500; Invitrogen-Molecular Probes, Darmstadt, Germany) for 1 h at room temperature. Cell nuclei were labeled with the nucleic acid dye 4’, 6-diamidino-2-phenylindole (DAPI, 300 nM; Invitrogen-Molecular Probes). In each case, secondary control incubations were performed to determine staining specificity.

The diaphragm was removed, then tumors were cut out and embedded into cryomatrix (Thermo Fisher Scientific, Waltham, MA, 6769006) as follows: Cryomolds (Tissue-tek Cryomold, VWR, Radnor, PA, 102094-040) were filled half full with cryomatrix and were frozen in isopentane (Sigma-Aldrich St. Louis, MO, 277258) chilled with liquid nitrogen. Cut-out diaphragms with tumors were put onto a teflon card and were frozen to ensure that the samples remain flat. The frozen samples were put on the top of the frozen cryomatrix and the mold was filled with cryomatrix and quickly frozen. Frozen sections cut perpendicular to the surface of the diaphragm (15 µm) were fixed in methanol (at −20 °C) for 10 minutes and incubated at room temperature for 1 hour with a mixture of primary antibodies (Table 1). After washing, sections were incubated for 30 min with appropriate secondary antibodies (Life Technologies, Carlsbad, CA) (Table 1). Samples were analyzed by confocal laser scanning microscopy using the Bio-Rad MRC-1024 system (Bio-Rad, Hercules, CA).