Zen 2

Histological brain sections were imaged using confocal microscopy (Zeiss inverted LSM800) or epifluorescence microscopy (Olympus VS120 Slide scanner). Confocal images were recorded on a Zeiss LSM 800 laser-scanning confocal microscope mounted with a plan-apochromat 10x/0.45, WD = 2.1 mm objective. Excitation/emission wavelengths were 488/509 nm (EGFP), 554/581 nm (tdTomato), and 353/465 nm (DAPI). Z-series images were collected on a PC running ZEN 2.6 software (Zeiss). Image series were Z-projected, stitched and contrast-enhanced using ZEN 2.6 software (Zeiss). Slidescanner images were recorded with a 10x/NA 0.4 objective. Excitation/emission wavelengths were 485/518 nm (FITCH), 560/580 nm (Cy3), and 387/455 nm (DAPI). Slide scanner images were processed using ImageJ software [70 (link)].

Serum-starved U2OS-R1 cells were incubated in DMEM supplemented with heparin (40U/mL) with FGF4-Fc (20 µg/mL) in the presence or absence of galectins (20 µg/mL) for 30 min on ice to assess cell binding and at 37 °C for 30 min to study FGF4-Fc endocytosis. Cells were subsequently washed with ice cold PBS, fixed in 4% paraformaldehyde solution and permeabilized with 0.1% Triton in PBS (for cells incubated at 37 °C). Nuclei were stained with NucBlue Live dye (Thermo Fisher Scientific), Zenon AF-488 (Thermo Fisher Scientific) was used for detection of FGF4-Fc. Early endosomes were detected with rabbit anti-human polyclonal early endosome antigen 1 (EEA1) antibody (#ab2900, Abcam) and anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (#A11037, Thermo Fisher Scientific). Wide-field fluorescence microscopy was carried out using a Zeiss Axio Observer Z1 fluorescence microscope (Zeiss, Oberkochen, Germany) as described in [33 ]. Images were processed with Zeiss ZEN 2.3 software (Zeiss, Oberkochen, Germany), and Adobe Photoshop CS6 (Adobe, San Jose, CA, USA). For quantification of the cell binding by FGF4-Fc in the presence of galectin variants, the total fluorescence of at least 20 cells from three fields of view/condition was measured in three independent experiments using Zeiss ZEN 2.3 software.

Specific antigen binding was determined by incubating sections with the respective primary antibodies for Cx43 (Cell Signalling Technology, Cambridge, UK), C3aR (Bioss, Woburn, MA, USA), and IL-1β (Abcam, Cambridge, UK) overnight at 4°C. A biotinylated IgG (Life Technologies, Carlsbad, CA, USA) was used as secondary antibody. Signal amplification was performed by using the VECTASTAIN® ABC Kit (Vector Laboratories, Burlingame, CA, USA), and signal was obtained by developing sections with the VECTOR® NovaRED™ Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA). Cell nuclei were counterstained in haematoxylin. Sections were investigated by bright-field microscopy using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Results are presented as mean pixel density. PAS staining was performed using a PAS-staining kit (Merck Millipore, Darmstadt, Germany). Signal density was measured using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Results are presented as mean density.

For analysis of cellular ROS, human CMs were seeded at a density of 6.3 × 10⁴ cells/cm² on ibidi 12-well slides (ibidi, Germany). Human CMs were treated with 100 ng/ml Midkine for 6 h at 37°C and 7% CO₂. After treatment, cells were incubated for another 30 min with 5 μM CellROX® Deep Red Reagent (Life Technologies, Carlsbad, CA, USA) at 37°C and 7% CO₂. Afterwards, cells were fixed with 4% formaldehyde and cell nuclei were stained with Hoechst. Cell were mounted with ProLong® Gold Antifade Mountant. Cells were investigated by blinded investigator by fluorescence microscopy using Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software. Imaging was performed by using 20x magnification (N.A. 0.5). Relative amount of reactive oxygen species was determined by Zeiss ZEN 2.3 software in order to exclude variations. For all experiments n = 6.

Proximity ligation assay (PLA) was performed using NaveniBright HRP kit (Navinci Diagnostics) according to the manufacturer’s protocol using the following primary antibodies: anti-cGAS (clone D1D3G, #15102, 1:100 pH9, Cell Signaling) and anti-DNA double stranded (clone AE-2, MAB1293, 1:100 pH9, Abcam). Negative controls were performed using only one primary antibody. Slides were analyzed under a Zeiss Axioscope A1 and microphotographs were collected using a Zeiss Axiocam 503 Color with the Zen 2.0 Software (Zeiss). Sections were subsequently immunostained to detect the expression of the E2.2 antigen. IHC was developed using SignalStainBoost IHC Detection rabbit (cod. #18653, Cell Signaling Technology) alkaline phosphatase-conjugated produced in horse and Vulcan Fast Red as substrate chromogen. Again, the sections were analyzed and photographed using Zeiss Axioscope A1 and Zeiss Axiocam 503 Color with the Zen 2.0 Software (Zeiss). Segmented images were obtained using HALO image analysis software (v3.2.1851.229, Indica Labs). HALO image analysis software was used to quantify the PLA signals in twelve non-overlapping fields at high-power magnification (400X) and the output was expressed as “percentage positive cells”.

Fluorescence signal was quantified from the original, unmodified TIFF file format images with Fiji software or from the original CZI file format with Zeiss Zen 2 software (Zeiss). In Fiji, sliding paraboloid algorithm was used to subtract background. The mean fluorescence intensity was quantified using an adequate threshold to select fluorescence signal and using the measure function. The brightness of the fluorescence signal has been adjusted in some images using Fiji or Adobe Photoshop (Adobe, San Jose, CA, USA), and all images presented together for comparison were adjusted similarly, unless otherwise stated. In Zeiss Zen 2, profile tab was used to measure fluorescence intensity profiles along a line intersecting the cytoplasm and the nucleus. Hoechst signal was used to determine the nuclear fluorescence. To be able to compare nuclear fluorescence intensity profiles in different cells and between different treatments the nuclear signal along the profiling line was set to start after the same distance. To normalize the fluorescence intensity profiles in control treatment the signal intensity at the starting point was set to 1. The mean intensity signal of the control treatment at the starting point was used to determine the relative fold change in the fluorescence profiles upon the treatment.

Lung and tumor tissues from experimental mice were harvested and embedded in OCT compound (Tissue-Tek, Torrance, CA). Cryosections (4-5 μm) were fixed in 100% acetone, followed by blocking with 3% bovine serum albumin (GenDEPOT, Barker, TX, USA) solution. The sections were stained with anti-CD3 (ab16669, Abcam), anti-CD4 (NBP1-19371, Novus Biologicals, CO, USA), anti-CD8 (ab4055, Abcam), anti-CD11c (ab11029, Abcam), anti-F4/80 (ab6640, Abcam), fibronectin (ab2413, Abcam) and vimentin (ab193555, Abcam) overnight at 4 °C. On the next day, sections were incubated with an Alexa Fluor^® 488- and 647-labeled secondary antibody (Abcam). Images of issues were acquired using a LSM880 confocal microscope (Zeiss, Jena, Germany) using the Zen 2.3 software (Zeiss) for image processing and quantitative analysis. The arithmetic mean of the optical intensities of the target proteins were measured in individual slide and normalized to those of DAPI (4′,6-diamidino-2-phenylindole) using the Zen 2.3 software (Zeiss, Jena, Germany). All experiments were performed in three randomly selected groups.