Zen blue 3

Histological slides were scanned using Nanozoomer digital slide scanner (Hamamatsu Photonics), Zen Blue 3.0 (Zeiss) and NDP view 2.0 (Hamamatsu Photonics) depending on the slide size. Scanned histological slides were then analysed and quantified using Fiji by Image J (National Institutes of Health, USA). Quantification of elastin and collagen fibers then proceeded using the colour deconvolution plugin, whilst collagen type immunohistochemistry proceeded with the immunohistochemistry (IHC toolbox) plugin in Image J v.1.53 (The University of Nottingham, UK). The process involved in the quantification of collagen and elastin fibers included the following steps; image acquisition, scale setting, RGB color space conversion, selection of the colour deconvolution toolbox, adjustment of the threshold value, measurement of the threshold area, quantification of the collagen or elastin fibers in the ROI, and imaging of the collagen and elastin fiber areas. Similarly, the process in quantification of collagen subtypes included; image acquisition, scale setting, RGB colour space conversion, selection of the IHC toolbox, adjustment of the threshold value, measurement of the threshold area, quantification of the collagen subtypes in the ROI, and imaging of the collagen areas. Each measurement was performed twice to minimize quantification errors.

Images were acquired with the confocal microscope Zeiss LSM900 equipped with Airyscan 2 – Super Resolution System and an oil immersion 63X objective (Zeiss, Zeiss Plan-Apochromat 63X/1.4 Oil) using the software Zeiss Efficient Navigation Blue (ZEN Blue 3.0, Zeiss). Once processed, images were analyzed with Fiji (ImageJ2 version 1.53c, National Institute of Health, USA). In the case of z-stacks, a z-projection was obtained averaging the fluorescence intensity. To select only nuclear condensates, measurements were performed inside nuclear ROIs based on DAPI staining of DNA. To remove the background and to ensure an analysis of true condensates, a threshold was automatically applied and particles with a surface lower than 20 px² were excluded. Resulting images were converted to masks and the Watershed Separation method was used to segment particles artificially fused during the z-projection. The circularity index of 1595 particles was analyzed.

Tissue clearing was performed using an adapted version of the ClearT2 method^{65 (link)}. Briefly, antibody labelled spheroids were incubated, with agitation, in 25% formamide and 10% polyethylene glycol 6000 (PEG) in water. After 10 min the solution was changed to 50% formamide, 20% PEG and incubated for 2 h replenishing every 30 min until spheroid opacity diminished. Spheroids were washed briefly in PBS before mounting on a microscope slide, with a 0.12 mm spacer, using ProLong Diamond Antifade Mountant with DAPI overnight. Spheroids were then imaged using a Zeiss LSM 800 confocal microscope using a 20 × objective. The aperture was set to 1 Airy unit and z-stack images were collected for each laser channel used (488 nm, 555 nm, 647 nm). Distances between z-stack image acquisitions were optimized per sample. Fluorescence and light microscopy were conducted using a Zeiss AxioVert A1 fitted with a Zeiss AxioCam digital camera. Images were analysed using Zeiss Zen Blue 3.0 software. Fluorescent signal from images was quantitated using the “Measure” tool built into the Zen Blue software package and normalised to relative fluorescence measurements to DAPI.

Three HAT1+/+ or HAT1−/− MEF cell lines were seeded in equal quantities on coverslips and allowed to attach for 24 h. Cells were then permeabilized with 0.5% Triton X-100 and fixed with 4% PFA simultaneously for 15 min, rinsed with PBS, and fixed again with 4% PFA for 10 min at room temperature. After several PBS washes cells were blocked with 5% BSA for 1 h at room temperature. BSA was removed with PBS. Nuclei were stained with 20 mM Hoechst 33342 Fluorescent Stain and mounted on slides using Vectashield. Images were acquired using a Zeiss LSM 900 Airyscan 2 Point Scanning Confocal microscope and Zen Blue 3.0. Quantification was completed using ImageJ version 1.52t by identifying nuclear regions, measuring the size, and recording nuclei that exhibited nuclear abnormalities.

Cells were visualized using an Axio Imager M2 optical microscope (Zeiss, Germany) in phase contrast and fluorescence mode. Photo documentation of the images was carried out using the Axoicam 506 Color camera and Zen Blue 3.1 (Zeiss, Germany). The effect of IBP on cell surface morphology and relief was studied using a combined scanning system consisting of an MFP-3D-BIO^TM atomic force microscope (AFM) (Asylum Research Inc., USA) and an Olympus Fluo View 1000 confocal laser microscope (CLSM) (Olympus Corporation, Japan). The differentiation between live and dead cells was performed with a LIVE/DEAD^®BacLight^TM Bacterial Viability Kit (Molecular Probes, USA). Preparation and scanning of samples were carried out following the previously described method by Ivshina et al. [21 (link)]. Root-mean-square roughness, length and width of cells were calculated from the height images. Cell volume and surface area were calculated using equations for cylindrical bodies [59 (link)]. The obtained images were processed using Igor Pro 6.22A (WaveMetrics, USA).

Concentrated antibodies were diluted, following optimal dilution found in previous tests, applied and incubated in a moist chamber (Table 2).
Immunomarked slides were digitalized using the slide scanner Axio Scan.Z1 (ZEISS, Jena, Germany). From the resulting digitalized file, between 10 and 15 images were generated using the software ZEN Blue 3.1 (ZEISS), representing the areas that adhered to the implant in the portion adjacent to the dermis (Fig. 1).
For quantification of the areas related to the immunohistochemical markers, the semiautomated color segmentation tool was employed, aided by the software Image-Pro Plus 4.5 (Media Cybernetics, Rockville, USA), with the purpose of determining the colors representing the immunoexpression and total tissue areas. The areas were artificially marked with red for immunoreactive regions and green for total tissue, aiming to standardize the value of the areas of structures of each image. Such color standardization was saved in a file named mask.
The mask was applied to all images, resulting in the area (square micrometers) of the two regions marked. The area values were exported to an Excel spreadsheet (Microsoft, Washington, USA).