Shandon cryomatrix

Following the completion of all in vivo measures, 8 animals were selected at random for immunohistological preparation. Rats were perfused transcardially with 200–300 mL of 0.1 M phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MA, USA) and fixed with 400–500 mL 4% paraformaldehyde (PF; Sigma-Aldrich, St. Louis, MA, USA). Lesion sites (±4 mm from epicenter; T1–T5 segments) were dissected following perfusion and stored in PF for no more than 48 h followed by at least 24 h in 10% sucrose before being flash frozen in Shandon Cryomatrix (Thermo Scientific, Cat: 67-690-06, Waltham, MA, USA) and stored at −80 °C.

Keratinocytes and dermal fibroblasts were seeded on collagen scaffolds (3 × 10⁵ and 2 × 10⁵, respectively) and incubated in complete medium at 37°C and 5% CO₂ atmosphere. The scaffolds were washed with PBS, fixed in 4% PFA, embedded in Shandon Cryomatrix (Thermo Scientific) and cryosectioned using a Leica cryotome in order to obtain 4 μm thick slices. The slices were subsequently subjected to haematoxylin‐eosin, DAPI, eosin‐Hoechst or Ayoub‐Shklar staining. For the first staining, the slices were incubated for 7 minutes with haematoxylin and 5 minutes with eosin Y, mounted in glycerol and visualized using a Zeiss Observer D1 microscope. For eosin‐Hoechst staining, after incubation (2 min) in eosin Y, the slices were differentiated with 70% ethanol, washed with distilled water, stained (10 min) with Hoechst (1 mg/ml), washed, mounted and visualized. For Ayoub‐Shklar staining, the slices were incubated with acid fuchsin 5% for 5 min, followed by 45 min in aniline blue‐orange G solution, washed and mounted. Additionally, keratinocytes and fibroblasts were stained with CellTracker^™ Red CMTPX Dye (Thermo Fisher Scientific), seeded on scaffolds and kept at 37°C and 5% CO₂ atmosphere. After 3 days, the scaffolds were washed with PBS and visualized by IVIS Spectrum CT System (Perkin Elmer, Caliper, LifeSciences).

High molecular weight dextran-FITC (500 KMW, Molecular Probes) was injected i.v. via the retro-orbital sinus (0.5 mg/0.1 mL) in mice bearing HEK-Lux or B16-Luc2 tumors. Harvested tumors were fixed in 4% paraformaldehyde (EMC) for 3–5 h at room temperature, depending on the tumor volume, followed by aldehydes quenching by 1 h incubation in 100 mM glycine (Sigma-Aldrich). Tumors were then incubated in 15% sucrose (Sigma-Aldrich) at 4°C overnight and in 30% sucrose at 4°C for ~24 h before embedding in Shandon Cryomatrix (Thermo Fischer) and freezing using isopentanol. Fifty micrometer sections cut using cryostat (CM3050 S, Leica) were stained with DAPI and imaged using an automated spinning disk microscope CellVoyager1000 (Yokogawa Electrics, Japan). The sections were left overnight at room temperature before being stained with DAPI.

Spheroids were generated from FaDu cells using the liquid overlay technique, as described previously [30 (link)]. Briefly, to form monoculture (F5) spheroids, 200 µL of FaDu cells (2.5 × 10⁴ cells/mL) was added to each well of a 96-well plate previously coated with 1% agarose (w/v in water) and cultured at 37 °C, 5% CO₂.
Co-culture (F5M5) spheroids were formed by seeding FaDu cells (100 µL at 5 × 10⁴ cells/mL) simultaneously with 100 µL of MeWo cells at 5 × 10⁴ cells/mL. To distinguish cells of different types in co-culture spheroid, MeWo cells were pre-stained with a membrane green fluorescent cell marker PKH67 (Sigma-Aldrich, Saint-Quentin Fallavier, France) according to the manufacturer’s recommendations, before seeding with FaDu cells.
At day 5 post-seeding, between 4 and 8 spheroids were used for each experimental condition. For immunohistochemistry analysis, spheroids were embedded into resin Shandon Cryomatrix (ThermoFisher, Waltham, MA, USA), frozen, cut and 10 µm thick sections were further analyzed by fluorescence microscopy. We used the cryosections with the diameter of the spheroid section about 400–450 μm.

Mouse embryos were collected from CO₂ euthanized pregnant dams and fixed with 4% paraformaldehyde (PFA) at 4°C overnight. For P7 studies, mice were lethally sedated with CO₂ and were transcardially perfused with 4% PFA. Samples were cryoprotected in 30% sucrose-PBS, embedded in Shandon Cryomatrix (Thermo Scientific), and sectioned using a Leica cryostat (CM 3050S).

Human colon biopsies and mice bowel samples were embedded into Shandon cryomatrix (Thermo Fisher Scientific) and cut into 5 μm slides, stored at -80 °C until use. HT-29 and CCD-18Co cells were seeded in chambers and cultured for 24 h in 37 °C. After repeated washing slides were permeabilized with Cytofix/Cytoperm (BD) for 15 min at RT, then incubated with primary antibodies specific to αSMA (sc-53015; mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at RT. After repeated washing slides were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen) and Alexa Fluor 568 donkey anti-rabbit secondary antibody (A10042, Invitrogen), both diluted to 1:100 for 30 min at RT in the dark and counterstained with Hoechst 33,342 (1:2000, Sigma-Aldrich). Finally, slides were rinsed in PBS and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).

BM was flushed out of femurs and tibias from female mice with Iscove’s Modified Dulbecco’s media (Gibco, Thermo Fisher Scientific, Ottawa, ON, Canada) containing 2% heat inactivated FBS, and supplemented with 2 mM l-glutamine, 50 µg/mL penicillin and 50 U/mL streptomycin. Recipient mice (Ldlr KO, 10–12 weeks old) were exposed to 1300 rad of ¹³⁷Cs irradiation using a Gammacell 3000 small animal irradiator (Best Theratronics, Ottawa, ON, Canada). BM (3 × 10⁶ cells/mouse) was injected intravenously via the tail vein. Mice were allowed to recover for 4 weeks, after which, atherosclerosis was induced by feeding a HF diet containing 21% butter fat and 0.15% cholesterol (catalogue number 112,286; Dyets Inc., Bethlehem, PA, USA) for 9 or 12 weeks. At the end of the feeding period, mice were fasted for 4 h prior to isoflurane anesthesia and euthanasia. Heparinized blood was collected by cardiac puncture and plasma was obtained by centrifugation at 4000 rpm in a microcentrifuge. Tissues were collected after in situ perfusion with 10 U/mL heparinized saline followed by 10% formalin, immersion fixed overnight in 10% formalin, and embedded in Shandon Cryomatrix (Thermo Fisher Scientific, Ottawa, ON, Canada). Plasma and tissues were stored at −80 °C until further analysis.