Mowiol mounting solution

In 24 well plates (Corning) with uncoated or 1µg/cm² fibronectin (Sigma) coated cover-slips (c.o.—Glaswarenfabrik Karl Hecht KG), 2.5 * 10³ MDA-MB-231 cells were seeded, in RPMI-1640 medium containing L-glutamate supplemented with 10% fetal bovine serum for 24h, 48h or 5 days. Cells were then fixed and permebabilized using methanol 80% / acetone 20% (stored at -20°C) for 10 min at RT (LAMP1 – ALIX) or 10 min PFA 4% fixation (0.04 g/mL – Merck), washed with PBS (3 × 10 min) and permeabilized 5 min with PBS-Triton 1% (Triton X-100—Carl Roth) (ITGA5 – SRSF6). Cells were washed with PBS-BSA 2% (Bovine serum albumin – VWR) and incubated with primary antibodies diluted in PBS-BSA 2% (Additional Table 4) overnight at 4°C in dark and humidified chamber. After being washed with PBS-BSA 2% (3 × 10 min), cells were incubated for 1 h at room temperature in dark with secondary antibodies, Hoechst (#H-21491 -Thermo Fisher Scientific) and probe (Additional Table 3). Cells were washed in PBS-BSA 2% and in PBS (2 × 5 min) and cover slips were mounted on microscope slides (VWR) with Mowiol mounting solution (Sigma-Aldrich) prewarmed at 56°C. Slides were kept at 4°C protected from light before the observation with the confocal laser scanning fluorescence microscope TCS SP5 (Leica).

Cells were grown on poly-D-lysine treated coverslips to reach 70% confluency the day of the experiments. After the treatments, cells were fixed with 4% paraformaldehyde in PBS supplemented with 0.1 mM CaCl₂, 0.1 mM MgCl₂ for 10 min at room temperature. Cells were washed three times with PBS before adding 50 mM NH₄Cl for 10 min at room temperature and then permeabilized with 50 μg/mL digitonin (Merck Millipore; D141) for 5 min at room temperature. Cells were then washed with PBS and blocked in 5% BSA (in PBS for 30 min at room temperature. Coverslips were incubated upside down with primary antibody in 1% BSA in PBS 1 h at room temperature, then washed three times with PBS and incubated upside down with secondary antibody in 1% BSA in PBS for 1 h. Finally, coverslips were washed three times in PBS and once with deionized water before mounting them on glass microscope slides using 10 µL Mowiol mounting solution (Millipore,475904). Fluorescence images were acquired using a Zeiss LSM 880 Airyscan confocal microscope with Plan-Apochromat 63×/1.4 Oil DIC M27 objective lens. Zeiss ZEN imaging software was used to acquire the images, and, after acquisition, processing was performed using an Airyscan processing tool on the ZEN software provided by Zeiss. All the antibodies used in this study are reported with the corresponding working concentrations in Table S1.

The tumors from the animals were embedded in paraffin and slides with 5μm thick tissue recuts were prepared. The tissue was deparaffinized and rehydrated prior to antigen retrieval in citrate sodium buffer (pH 6.0) for 20 minutes. The sections were incubated with primary antibody for one hour at room temperature, followed by one-hour incubation with a secondary antibody labeled with DyLight 594 or 488 (Vector Laboratories, Burlingame, CA, USA). The nuclei were labeled with NucBlue Live ReadyProbes Reagent (Life Technologies Carlsbad, CA, USA) for 10 minutes and mounted with Mowiol mounting solution (Millipore, Billerica, MA, USA). The staining was analyzed by fluorescence microscope (Zeiss, Oberkochen, Germany). The primary antibodies used included anti-Ki67 (ab15580) and anti-CD31 (ab28364) (Abcam, Cambridge, UK). When the tissue was co-stained to indicate hypoxic regions, a 30-minute incubation step with FITC-MAb1 (Hydroxyprobe, Inc, Burlington, MA, USA) at room temperature was added after the staining with secondary antibody. FITC-MAb1 binds to adducts formed by hypoxia-activated pimonidazole with thiol groups in proteins, peptides, and amino acids.