Bz x analysis software

Discernable MHC I, MHC IIa, and MHC IIx fibers within the ×20 field of view were traced and quantified on the accompanied autofluorescence and mitochondrial content-stained image using BZ-X Analysis Software (Keyence). Myofibers positive for MHC I and negative for MHC IIa were classified as type I, fibers positive for MHC IIa and negative for MHC I were classified as type IIa, and fibers negative for both MHC I and MHC IIa were classified as type IIx. Myofibers coexpressing more than one MHC isoform were classified as hybrid fibers and excluded from analyses. The number of myofibers included in analyses were 65 ± 10. Nuclei density was assessed by computational counts of DAPI-stained nuclei. Muscle fiber type and nuclei characteristics are presented in Table 3. For measurable comparisons, fiber-specific NADH and Fp signals were standardized to the same exposure time, instrument gain, and light source intensity. Exposure time was optimized so that cytosolic compartments were free of saturation. Background signal intensity was subtracted from all fluorescence imaging signal intensities when calculating relative intensities specific to each fiber type.

Formalin-fixed, paraffin-embedded tumor tissues were cut into serial 4 μm sections and immunostained with anti-vimentin antibodies to evaluate stromal volume in human CRC. After vimentin immunostaining, the proportion of vimentin-positive areas in the CRC was evaluated. The correlation between the number of vimentin-positive regions and LAT1 expression in CRC was also examined. Observations were made using a BZ-X710 all-in-one fluorescence microscope (KEYENCE, Osaka, Japan). Microscopic fields that included the foci were photographed at 400× magnification for each specimen and analyzed. All the micrographs were obtained under the same conditions (exposure time, gain, illumination light intensity, and aperture stop). The vimentin-positive areas were determined by arranging and quantifying the brightness thresholds using the BZ-H3C hybrid cell count application of the BZ-X analysis software, version 1.3.1.1 (KEYENCE).

We conducted a histological analysis of lung injury according to previously described methods [22 ]. Briefly, lung tissues were collected from anesthetized mice 1 h after histone injection (60 μg·g⁻¹) and weighed, after which the tissue samples were embedded in OCT compound (Sakura Fine Technical, Tokyo, Japan) and frozen in liquid nitrogen. Thereafter, lung tissues (10 μm thick) were prepared using a microtome and stained with hematoxylin and eosin. Whole section images were captured at 40× magnification using a BZ‐X800 fluorescence microscope (Keyence, Osaka, Japan). To evaluate the extent of pulmonary hemorrhaging, we measured the proportion of the bleeding area using bz‐x analysis software (Keyence).

Polyclonal goat anti-Lcn2 (AF1857; R&D systems), monoclonal mouse anti-neuron specific enolase (NSE; BBS/NC/VI-H14; Dako, Glostrup, Denmark), monoclonal mouse anti-glial fibrillary acidic protein (GFAP; G-A-5; Sigma-Aldrich) and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (G3pdh; Medical and Biological Laboratories, Aichi, Japan) antibodies were the primary antibodies used in this study. Peroxidase-conjugated antibodies (GE Healthcare, IL, United States) or fluorescent-conjugated antibodies (Jackson ImmunoResearch, PA, United States) were used as secondary antibodies for immunoblotting or immunohistochemistry, respectively. Immunoblotting, TdT-mediated dUTP nick and labeling (TUNEL) assay, and immunohistochemical analysis of Lcn2 were carried out as described previously (Ueno et al., 2018 (link)). For the immunohistochemical analysis of GFAP, paraformaldehyde-fixed retinae were immersed in increasing concentrations of sucrose (10–30%) and embedded in frozen medium; cryosections (5 μm thickness) were further treated with 0.1% Triton X-100. TUNEL positivity, GFAP immunoreactivity, and nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories, Kumamoto, Japan) were visualized with a BZ-X710 microscope (Keyence, Osaka, Japan) and measured using the BZ-X analysis software (Keyence).

For localization of the CLR protein in cryo-embedded spine samples, nonconsecutive serial sections were used. For staining of CLR sections, sections were permeabilized with PBS/0.5% Triton X-100 for 10 min and washed with PBS/0.25% Triton prior to blocking in 3% BSA/5% donkey serum with 0.1% Triton. After blocking, the sections were incubated overnight with an anti-CLR primary antibody (rabbit) (1:200, bs-1860R-TR, Bioss Antibodies). Next, the sections were washed in PBS, incubated with the secondary antibody (1:400, anti-rabbit-Cy3, 711-165-152, Dianova) and mounted in Fluoromount-G with DAPI (Thermo Fisher). Images were acquired using a Keyence BZ-X810 microscope (Keyence) and Keyence BZ-X Analysis software (Keyence).

Formalin-fixed kidneys were transferred to 80% ethanol and embedded in paraffin. Three µm sections were cut and stained with hematoxylin/eosin (Histology Core, Lerner Research Institute) or Periodic acid shiff (PAS) (Thermo Scientific, Waltham, MA, USA) for detection of kidney morphology, damage and cellular infiltrates. Sections were scored in a blinded fashion by a renal pathologist (JN) at the Cleveland Clinic. Kidneys were evaluated on a scale of 0–5 for mesangial and endothelium hypercellularity. Glomerular area was calculated by measuring the area of 5–15 individual glomeruli per section for each mouse. Immunofluorescence was performed on 5µm sections of kidneys preserved in OCT™ (Scigen, Paramount, CA, USA). Sections were thawed, fixed in acetone, blocked with 10% normal goat serum, and stained using Texas-Red conjugated anti-IgG or anti-IgM antibodies (SouthernBiotech, Birmingham, AL, USA) and FITC-conjugated anti- Complement factor C′3 antibodies (Immunology Consultants Laboratory Inc., Portland, OR, USA). Imaging was done on a Keyence BZ-X700 All-in-one microscope (Keyence, Osaka, Osaka, Japan) and images were quantified using the Keyence BZ-X analysis software (Keyence, Osaka, Osaka, Japan). A total of 5–15 glomeruli were quantified for each section and the average calculated per mouse.

Half kidneys and 2mm cross sections of spleens were isolated and immediately frozen in OCT™. Five µm sections were cut and sections were stained for the presence of IgG and complement C’3, or B220 and GL7, respectively. Briefly, sections were fixed with cold acetone and blocked with unlabeled anti-mouse CD16/CD32 (1:200, EBiosciences) in 10% non-immune goat serum (Invitrogen). Texas-red conjugated anti-mouse IgG (1:500, Southern Biotech), FITC-conjugated anti-mouse C’3 (1:500, ICL), FITC-conjugated anti-B220 antibodies (EBiosciences), biotinylated anti-GL7 antibodies (eBioscience), and Alexa Fluor 568–conjugated streptavidin (Invitrogen) were added as indicated for each staining combination and sections were incubated overnight at room temperature. The next day, sections were washed and mounted with 70% glycerol. Imaging was done on a Keyence BZ-X700 All-in-one microscope (Keyence, Osaka, Japan) and images were quantified using the Keyence BZ-X analysis software (Keyence, Osaka, Japan). Colocalization of IgG/C’3 in renal samples is displayed by the color yellow.