Tcs sp5 aobs

RNase3, RNase6, and RNase7 were labeled with Alexa Fluor 488 Labeling kit (Invitrogen, A10235), following the manufacturer′s instruction as previously described (21 (link)). To 0.5 mL of 2 mg/mL protein solution in PBS, 50 μL of 1 M sodium bicarbonate, pH 8.3, was added. The protein was incubated for 1 h at room temperature with the reactive dye, with stirring, and the labeled protein was separated from the free dye by PD-10 desalting column (GE Healthcare, 17-0851-01). Labeled protein distribution in cell cultures was followed by confocal microscopy. About 2.5 × 10⁵ RAW cells were harvested in 3 cm diameter microscopy plates (MatTek, P35G-1.5-14-C) 2–3 h before the assay. Macrophages were washed with RPMI and labeled with Hoescht 33342 (Thermo Fisher Scientific, 62249) and Cell Mask Deep Red Plasma membrane Stain (Thermo Fisher Scientific, C10046) at 0.5 μg/mL for 5–10 min before observation in Leica TCS SP5 AOBS equipped with a PL APO 63 × 1.4–0.6 CS oil immersion objective (Leica Microsystems, Mannheim, Germany). Following, Alexa Fluor labeled proteins were added at 2 μM to the cultures and time lapse was recorded at intervals of 30 s for 30 min. Fluorochromes were excited by 405 nm (Hoechst 33342), 649 nm (CellMask Deep Red), and 488 nm (Alexa Fluor 488). Emissions were collected with a HyD detector.

Widefield fluorescence microscopy images of hTCEpi cells were collected on a Nikon Inverted TE2000i (Nikon Corporation, Tokyo, Japan) microscope using a 20×/0.50 Plan FLN objective, Cascade II camera (Photometrics, Tuscon, AZ, USA) and controlled using NIS-elements software (Nikon Corporation, Tokyo, Japan). Confocal images were collected on a Leica TCS SP5 AOBS (Leica Microsystems, Wetzlar, Germany) upright confocal using a 63×/0.90 water-dipping objective and 3× zoom. The confocal settings were as follows, pinhole 1 airy unit, scan speed 400 Hz unidirectional. Images were acquired using sequential scanning and appropriate laser and filter settings for each channel to ensure that there was no bleed through between fluorescent channels. The images were collected sequentially to eliminate cross-talk between channels. An optical section was acquired every 1 µm. Images were processed with Image J software or Leica Application Suite Advanced Fluorescence software (version 3.1.0) (Leica Microsystems, Wetzlar, Germany). Only the orthogonal views of these 3D stacks are shown in the results.

The metabolic activity of embedded cells was assessed using the resazurin assay through incubation of cell-laden gels in DMEM medium containing 20% v/v resazurin sodium salt (Sigma-Aldrich) for 2 h at 37 °C. Samples were measured using a microplate reader (Synergy MX, Biotek, Winooski, VT, USA) at 530 nm (excitation) and 590 nm (emission). To evaluate cell morphology and fibronectin deposition in the dermis, cell-laden hydrogels were fixed in 4% v/v paraformaldehyde (PFA, Electron Microscopy Sciences) in Hanks’ Balanced Salt Solution (HBSS, Life Technologies, Carlsbad, CA, USA) for 30 min, followed by HBSS washing. Samples were permeabilized with Triton X-100 (0.1% v/v, 10 min, Sigma-Aldrich) in HBSS and were washed and incubated in blocking solution (1% w/v bovine serum albumin, BSA, Irving, TX, USA) for 1 h. Then, samples were incubated overnight at 4 °C with phalloidin/Alexa Fluor 488 (1:40, Molecular Probes-Invitrogen, Eugene, OR, USA) and rabbit anti-fibronectin antibody (1:400, F3648, Sigma-Aldrich). Afterward, samples were rinsed with HBSS and incubated for 45 min with Alexa Fluor 594 goat anti-rabbit secondary antibody (Molecular Probes-Invitrogen) and Hoechst for nuclei staining. Finally, samples were rinsed and confocal images were acquired using a laser scanning microscope (CLSM, Leica TCS-SP5 AOBS, Leica Microsystems, Wetzlar, Germany).

After treatment and washing, carried out as described in Section 2.6., cells were stained with CellMask™ Orange Plasma Membrane Stain (dilution 1:20 in PBS) for 5 min at 37 °C, fixed with PFA 2% for 15 min, and then stained with DAPI (100 ng/mL) 15 min at room temperature in the dark. A double washing with PBS followed each passage. At the end, coverslips were mounted onto clean slides and maintained at 4 °C in the dark. The analysis was made by Spectral Confocal Microscope Leica TCS-SP5 AOBS, Leica Microsystems (Wetzlar, Germany) using a 63× HCX PL APO CS oil objective and performing Z stacks (zoom of 1.0). Images were acquired by setting the pinhole at 95.5 μm, the image size of 512 × 512 pixels, 8 bits of resolution and a bidirectional scanning speed at 400 Hz. DAPI, FITC-labeled NPs, and the CellMask™ Orange Plasma Membrane Stain were excited by a 405 Diode UV, an argon, and a DPSS 561 laser, respectively. Untreated cells were used as a control to adjust the gain of each detector and remove the green autofluorescence of the cells. Using the same adjusted settings, a Z-stack of all treated samples was performed with a step size of 0.5 µm (20–30 slices per each image). Images were treated through ImageJ 1.48 V Software.

Immunofluorescence analysis on tumor cells and tissues was performed as described [23 (link),24 (link)]. Staining was carried out with the following primary antibodies: anti-αSMA (ab5694), anti-TNC (ab108930), and anti-Collagen-I (ab34710) all from Abcam (Cambridge, UK), and revealed by Alexa Fluor 488-conjugated secondary antibody. All images were captured with a Leica TCS SP5 AOBS confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany). Immunofluorescence acquisition settings were kept constant within each cell line or tumor tissue. Mean fluorescence intensity (MFI) was evaluated with ImageJ software, measuring the mean pixel intensity in each channel, background subtracted. MFI was normalized on DAPI tumors.