Zen 2.3 lite

The confocal images (Fig. S2B, top panel) of zebrafish intestine stained with LysoSensor and Neutral Red were taken with a Zeiss LSM 880 confocal microscope using 20× air lens (NA=0.8). The zoomed-in images (Fig. S2B, bottom panel) were generated from the non-saturated area in intestine of the original images using Zen 2.3 Lite (Zeiss). Colocalization analysis of both LysoSensor- and Neutral-Red-positive cells was performed using ImageJ Fiji software. The 2D intensity histogram and Pearson correlation coefficient were generated by the Coloc2 plugin of Fiji.

Fluorescent binding assays using fluorescently tagged RBP1_Phi4.2 and RBP2_Phi4.2 were performed as described previously [41 (link)]. Briefly, 0.3 mL of L. lactis 4 in the exponential growth phase (taken as an OD_600nm of between 0.4 and 0.6) was harvested and resuspended in 100 µL of SM buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgSO₄). Cells were incubated with either 5 µg/mL of mCherry-RBP1_Phi4.2 (the maximum quantity possible due to poor expression) or 50 µg/mL of mCherry-RBP2_Phi4.2 for 12.5 min at 30 °C. Cells were washed twice in SM buffer, and fluorescent binding was visualized by confocal microscopy (Zeiss LSM 5 Exciter, Zeiss, Germany) to achieve high-resolution images, with a wavelength of 514 nm for mCherry. Strain L. lactis 10, isolated from the same facility as L. lactis 4 [27 (link)], was used as a negative control. Images were analysed and compiled using the Zen 2.3 Lite software package (Zeiss, Oberkochen, Germany).

Cancer cells were live stained with PKH67 cytotracker-green (Sigma-Aldrich, USA) before seeding into the device. After extravasation, the ovarian cancer cells were incubated at 37°C with continuous perfusion of medium in tumor chamber for 3 days. Invasion dynamics of live-stained cancer cells (green) into the chip ECM chamber hydrogel was visualized using fluorescence microscopy (ZEISS Axio Observer; LD Plan Neofluar 10×, numerical aperture 0.4). Snapshots were taken at every 12 hours of interval with an exposure time of 200 ms. Images were analyzed and processed using software ZEN 2.3 lite (ZEISS). Measurement of cancer cell invasion through the hydrogel was performed using cell counter plugins in ImageJ. The ECM invasion of cancer cells was calculated as the ratio of area occupied by invaded cancer cells in ECM chamber versus the total area of ECM chamber (55 (link)).

Prior to sectioning, the deeply frozen brain samples were allowed to warm at −11 °C in a Leica cryostat CM1950 (Wetzlar, Germany) chamber. The striatal level was located using a stereotaxic atlas²⁸ . In the MALDI MSI, LA-ICP-MSI and histology analyses, consecutive sections of 10-, 30- and 30-µm width were used, respectively. The sections were thaw-mounted onto precooled ITO glass slides for the MALDI MSI and light microscopy analyses. For the LA-ICP-MS analyses, the sections were directly deposited onto an Omni Slide without thawing. All slides were vacuum-dried in a desiccator at room temperature for 40 minutes prior to further use.
For histology evaluation, the slices were rehydrated in descending aqueous ethanol concentrations (100%, 95% and 80%) and stained with haematoxylin, Gill No. 2 (Sigma Aldrich, Prague, Czech Republic) to visualize the cellular nuclei. Then, the slide was flushed with water, and the excessive colorant was removed by acidic ethanol. Eosin counterstaining was subsequently performed. Tissue slices were serially dehydrated in ethanol solutions (95% and 100%), toned with xylene and permanently fixed with DPX mounting medium. Finally, the slides were examined as light-microscopic images using an Axio Scan.Z1 slide scanner (Zeiss, Jena, Germany) and processed using ZEN 2.3 Lite (Zeiss, Jena, Germany) software.

Two cell lines, i.e., 5-8F (a CAIX positive control [31 (link)]) and C666-1 (a CAIX negative control [32 (link)]) were provided by Southern Medical University. The cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum and with penicillin and streptomycin (Gibco Invitrogen, Carlsbad, CA, USA). The cells were incubated on confocal plates (2 × 10⁵ cells/plate) for 24 h. After removal of the medium, CAIX-FITC and free FITC were incubated separately with the cells for 4 h at a final concentration of 10 nM. The cells were then washed with phosphate-buffered saline three times and fixed with 4% paraformaldehyde for 15 min at 37 °C. The cytoskeleton was first stained with rhodamine phalloidin for 30 min and the nucleus was stained with 15 μg/ml of DAPI (4, 6-diamidino-2-phenylindole) for another 8 min at room temperature. All images were acquired using a confocal laser scanning microscope (LSM-710, Carl Zeiss, Oberkochen, Germany). Imaging processing was performed using ZEN 2.3 lite (Zeiss, Germany). Fluorescence quantification was analyzed using ImageJ 2.X (LOCI, University of Wisconsin).

Gastrocnemius muscles were quickly dissected, mounted in 9% Tragacanth gum (Sigma, G1128), frozen in liquid nitrogen-cooled isopentane, and kept at -80 °C. 10 µM cryosections were blocked with mouse on mouse (M.O.MTM) blocking reagent (MKB-2213, Vector Laboratories) before overnight incubation at 4 °C with anti-laminin (L9393, Sigma) primary antibody in DPBS buffer supplemented with 0.5% BSA (A7030, Sigma). The next day, slides were washed in DPBS and stained with anti-rabbit Far Red-Alexa Fluor 647 (711-175-152, Jackson ImmunoResearch) secondary antibody, in DPBS buffer supplemented with 0.5% BSA, for 1 h at 37 °C. After washing in DPBS, slides were mounted in Fluoromount G medium (FP-483331, Interchim, Montluçon, France). Whole muscle section images were acquired at a 10x magnification with a wide-field fluorescence video-microscope (Video Microscope Cell Observer, ZEISS, Oberkochen, Germany). Mosaics were then stitched (Zen 2.3 lite, Zeiss). For each sample, altered fibers were manually removed. Segmentation of all muscle fibers from a cryosection was performed using Cellpose^{39 (link),40 (link)}. Mean fiber cross-sectional area was obtained using a self-developed Python script using all muscle fibers from each section.