To analyse the structural characteristics of collagen fibres in each region, a variety of measurements were obtained using Imaris v9.6.0 (Oxford Instruments) and OrientationJ83 (link) (http://bigwww.epfl.ch/demo/orientation/ ) and GLCM (https://imageJ.nih.gov/ij/plugins/texture.html ) plugins for (Fiji/ ImageJ). Imaris v9.6.0 (Oxford Instruments) surface tool was used to segment collagen fibres and tissue autofluorescence to measure collagen intensity and density (defined as % of tissue covered with collagen), and to quantify the number of KRT5+ cells in the different analysed regions. OrientationJ was used to measure fibre coherency utilising properties of the original image, so-called ‘first-order statistics’. Grey-level co-occurrence matrix (GLCM) texture analysis derives statistical measures by considering the spatial relationship of pixels in an image, so-called ‘second-order statistics’. The GLCM features measured included contrast, entropy, correlation, inverse difference moment (IDM) and angular second moment (ASM) (Supplementary Table S2 ).
Imaris v9
Manufactured by Oxford Instruments
Sourced in United Kingdom, Switzerland
Imaris v9.5 is a powerful 3D/4D image analysis software suite developed by Bitplane, a subsidiary of Oxford Instruments. The software provides a comprehensive suite of tools for visualizing, processing, and analyzing complex microscopy data, including confocal, multiphoton, and super-resolution imaging.
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Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Lsm780 confocal laser scanning microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rnascope 2.5 hd assay, by Advanced Cell Diagnostics (2 mentions)
Eclipse ni microscope, by Nikon (2 mentions)
Ultramicroscope blaze, by Miltenyi Biotec (2 mentions)
Vari mix platform rocker, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Bio-Techne (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Lsm 900, by Zeiss (1 mentions)
Airyscan super resolution microscope, by Zeiss (1 mentions)
Axio imager 2 light microscope, by Zeiss (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Lightsheet microscope, by Zeiss (1 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Triton x, by Merck Group (1 mentions)
Ihc tek diluent, by IHC World (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
21 protocols using imaris v9
1
Collagen Fiber Structural Analysis
2
Automated 3D Brain Reconstruction
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Image analysis and 3D reconstructions were performed using Imaris v9.5 software (Bitplane, Oxford Instruments) after removing autofluoresence using the Imaris Background Subtraction function with the default filter width so that only broad intensity variations were eliminated. Additionally, the entire brain was defined as an ROI in order to mask all background fluorescence outside the spinal cord surface. Artifact and nonspecific fluorescence surrounding the brain were segmented and removed using the automatic isosurface creation wizard based upon absolute intensity. Voxels contained within the created surface were set to zero, and the remaining mask was used for all further analysis. Automatic segmentation of nuclei within specified ROIs was applied using the spots detection function and later superimposed on a maximum intensity projection volume rendering of the tissue. For some of the figures, surfaces were created around the brains and spinal cords to make them more evident in the 3D reconstructions. Quality thresholds were set based upon visual inspection of the mixed model rendering for both spots and surfaces.
3
Visualizing Platelet-Fibrin Clot Formation
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To prepare PRP clots for visualization, 100 μl of PRP was incubated with AF647-conjugated CD42b rabbit anti-human antibody (1 μg/ml; catalog no. 303923, BioLegend, San Diego, CA, USA) to label the platelets, and 1% AF488-conjugated fibrinogen (catalog no. F13191, BioLegend, San Diego, CA, USA) to label fibrinogen for 10 min on a rocker with gentle rocking frequency of approximately 2 Hz (Vari-mix Platform Rocker, Thermo Fisher Scientific, Waltham, MA, USA). After incubation, clots were prepared by adding 20 mM CaCl2 and thrombin (0.2 U/ml). Immediately, 50 μl of the mixture was added on ethanol-cleaned 1-mm microscope slides (Thermo Fisher Scientific, Waltham, MA, USA). PPP clots were prepared in a similar fashion but without the platelet antibody. The clots were formed at 22°C for 45 min. The clots were sandwiched between coverslip and slide and imaged by confocal microscopy with an Apochromat 63× oil objective with a vertical stack interval of 1 μm (Zeiss LSM 700) for quantitative analyses. The images were analyzed using Imaris v9.5 (Oxford Instruments, MA) and National Institutes of Health ImageJ (93 (link)).
4
Biofilm Architecture Quantification
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To quantify biofilm architecture, the confocal images were analyzed using Imaris v9.5 (Oxford Instruments, MA). The average intensities of SYTO 9 and SYPRO ruby in every image of the z-stack were obtained using an in-house MATLAB script (https://github.com/tcheng1124/Matlab.git ) at various time points. These intensity values were then used to calculate the growth of the structure over time after applying a minimum threshold value. In addition, the distributions of microcolony sizes and volume colocalization statistics were obtained using the Imaris image-processing module. The microcolony size distribution was obtained using the surface-rendering feature using the green channel and a minimum volume threshold of 2.00 μm3. The Pearson correlation coefficient (PCC) was estimated using a built-in function in Imaris software.
5
Whole-Liver Perfusion and Microscopy
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The livers of freshly-sacrificed mice were perfused retrogradely via the IVC(3) with 3 ml of PBS and then 10 ml of 2% paraformaldehyde (Sigma, catalogue# 30525-89-4) in PBS. The gallbladder was removed and the liver was fixed in 2% paraformaldehyde in PBS for 8 hours. A section of the liver was embedded in 3% agarose (Fisher Biotec, catalogue# AGR-LM-50) and 150μm thick sections were cut using a Vibratome 1000 Plus Sectioning System (Harvard Apparatus, Holliston MA). Sections were blocked with 4% bovine serum albumin (Tocris bioscience, catalogue# 9048-46-8), 5% normal goat serum (Invitrogen, catalogue# 31873) and 0.3% Triton-X 100 (Sigma, catalogue# 9002-93-1) in PBS for 20 hours at 4°C. Sections were stained with primary antibodies for 20 hours at 4°C, washed, then incubated with secondary antibodies for 20 hours at 4°C, followed by staining with DAPI (Sigma, catalogue# 28718-90-3) for 1 hour at 4°C. Primary and secondary antibodies were diluted in blocking buffer. Washing buffer comprised 0.1% Triton-X 100 in PBS. Images were acquired using a Leica SP8 confocal microscope at 93x objective magnification with a numerical aperture of 1.35. The images were analysed using Imaris v9.5 (Oxford instruments).
6
Detailed Dendritic Spine Imaging Protocol
7
Detailed Microscopy Protocols for Dendritic Spines
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With EM gain and no binning, the acquisition was set to 10 MHz, 14 bits. The EM gain multiplier restrained below 300, and auto exposure was set between 100–300 ms and. Within the first quarter of the scale (<4000), the laser power was adjusted to keep LUTs. Imaging and reconstruction parameters were determined with the assistance of the expertise in the Center for Advanced Microscopy at Northwestern University. The single-plane where the spine head was in focus, based on the cell fill, was chosen for analysis. Each spine head was outlined using the Image J software manually in the channel of the cell fill to detect the area. Within the spine head, ankyrin-G and Homer1b/c puncta were outlined manually, and the size was recorded. A 100 μm dendritic region was selected, and puncta counts were made; puncta smaller than 0.006 μm2 were excluded from the analysis. To present more informative images, z-series images were reconstituted by Imaris v9.1.2 (OXFORD instruments).
8
Quantifying Viral Transsynaptic Tracing
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Spinal cord tissue for these analyses was collected at 30 WPI and sectioned horizontally at 30μm thickness using CryoJane tape transfer method. Immunostaining for ⅙ sampling interval sections was performed to detect PRV-GFP labeled fibers. For quantification, five to six random optical fields within the lesion site per section were imaged using Zeiss LSM 900 with Airyscan super resolution microscope. Antibody source and the dilutions were used as listed in Supplemental Table 1. For each optical field, 24 μm Z stack images (0.4 μm Z-step) were captured using 60X oil objective. PRV-GFP labeled filament volumes were manually traced using Imaris v9.6 filament manual tracing software (Oxford Instruments, Abingdon, United Kingdom ). Brain sections were sectioned coronally at 30μm using a sliding microtome. Immunostaining (all sections containing motor cortex, no sampling) was performed to detect PRV-GFP cell bodies. All PRV+ cell bodies within the motor cortex were counted manually. Quantification was performed at 20X magnification using ZEISS Axio Imager II light microscope with an Apotome2 image processor. Image acquisition and quantifications were performed by investigators blinded to the experimental groups.
9
3D Analysis of Cellular Stress Granules
10
Quantifying Neutrophil Extracellular Traps
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NET formation was tested using confocal microscopy as previously described (79 (link)). Sorted CD16Int (0.5 × 106 cells/well) were resuspended in NET media (colorless RPMI + 0.5% BSA +10 mM HEPES) and seeded onto sterile acid–washed coverslip coated with (1 mg/mL) poly-l -lysine. Cells were incubated for 60 minutes in CO2 incubator. After incubation, cells were fixed with 2% PFA for 30 minutes, washed twice with PBS, and blocked with 1% BSA in PBS for 1 hour. NETs were determined by extracellular colocalization of anti-human lactoferrin antibody (1:500 dilution, MP Biomedicals, catalog 55040) with DAPI (600 nM for 10 minutes) nuclear stain. The secondary antibody utilized was Alexa Fluor 647 (1:1000; Invitrogen, Thermo Fisher Scientific, catalog A21246). Confocal images and Z-stacks (1 μm thickness for each slice) were obtained by the Fluoview FV1000 confocal microscope (Olympus) with the 63× oil objective. Confocal Z-stack images were used to quantify colocalization of extracellular DNA and lactoferrin using IMARIS v9.6 software (Oxford Instruments).
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