Image pro analyzer 7

The skeletal phenotype of BgnFmod KO mice was first analyzed in whole femurs and L3 lumbar vertebrae using the µCT50 system (Scanco Medical AG, Brüttisellen, Switzerland) at a 10-μm resolution. Mineralized tissues were segmented by a global thresholding software. Standardized nomenclature was used for the bone parameters measured. For the femurs, trabecular parameters were measured at the secondary spongiosa of the distal metaphysis and cortical parameters were determined in a 1mm ring at the mid-diaphyseal region according to previously published guidelines^{88 (link)}. For the 3^rd lumbar vertebra only trabecular parameters were measured. Growth plate thickness was measured in the distal epiphysis of 5-week old mice. Briefly, for each sample, 2D projections from anterior, posterior and mid-longitudinal locations were obtained, the average thickness of the growth plate obtained from the three projections was quantified using the ImagePro Analyzer 7.0 software (Media Cybernetics, Inc.).

Detection of protein oxidation by reactive oxygen species (ROS) was measured using the Protein Carbonyls Immunohistochemical Staining Kit (Cosmo Bio USA, Carlsbad, CA, USA, Catalog No. SML-ROIK04-EX). A goat, anti-rabbit IgG primary antibody (Vectastain Elite ABC HRP Immunodetection Kit, Novus Biologicals, LLC, Centennial, CO, USA, Catalog No. PK-6101-NB) was applied on each sample. Secondary solutions A and B (Vectastain Elite ABC HRP Immunodetection Kit) were subsequently applied. Slides were developed using HRP Color Development Reagent, DAB (Bio-Rad Laboratories, Hercules, CA, USA, Catalog No. 170-6535) and counterstained with hematoxylin (Fisher Scientific, Hampton, NH, USA, Catalog No. ICN10192225).
Photographs of whole tissue slides were taken at 20× magnification using a VS120 Virtual Slide Microscope (Olympus, Tokyo, Japan). Images were converted from .vsi to .tif and compressed using the Fiji distribution of ImageJ 1.52p (Wayne Rasband, National Institutes of Health, USA) [57 (link)]. Percent positive staining was determined as the number of positive shaded pixels (brown) divided by the total number of pixels representing the whole tissue multiplied by 100. All pixel analysis was performed using Image-Pro Analyzer 7.0 Software (Media Cybernetics, Rockville, MD, USA).

Double stains for VACV and the marker of interest were performed for each chromogenic IHC stain. H-score quantitation for each of the protein markers was performed by one in-house pathologist by counting a total of 100 cells using the predetermined ROIs from lesioned and adjacent MPXV–IHC-negative skin. The number of positively stained cells was measured as the percentage of the total numbers of counted cells and expressed as percent-positive cells. The staining intensity for each marker was visually scored 0–3 (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining) resulting in H-scores of percent-positive cells that ranged from 0 (no staining) to 300 (diffuse, intense staining) [41 (link)]. Manual cell counts were performed at 20× magnification.
To reduce visual bias during manual cells counts (H-scoring), a calibrated, computerized grid composed of squares measuring 20 µm across was applied to the ROIs using Image Pro Analyzer 7.0 software (Media Cybernetics, Rockville, MD, USA). Cells were counted from the left of the preselected ROIs and continued to the right following the horizontal lines of the computerized grid until a total count of 100 was reached. To further reduce visual counting bias, only those cells whose nuclei were overlaid by cross hatches of the computerized grid were counted (Figure 1A).

Prior to sacrifice WT and BgnFmod KO mice were injected with a calcein fluorochrome (Sigma-Aldrich, St. Louis, MO), 15 mg/Kg intraperitoneally. 5-week old mice were injected with calcein 4 days and 1 day prior to sacrifice while 78-week old mice were injected with calcein 12 days and 1 day prior to sacrifice. Following µCT scanning the femurs were embedded undecalcified in methyl methacrylate. Mid-frontal longitudinal sections were prepared. For dynamic histomorphometry based on the vital calcein double labeling the sections were left unstained. To identify osteoclasts, consecutive sections were stained for TRAP (294–67001, Wako Pure Chemical Industries, Osaka, Japan). Quantitation analysis for both assays was performed using ImagePro Analyzer 7.0 (Media Cybernetics, Inc.). The bone formation and resorption parameters were determined in the secondary spongiosa of the distal metaphysis as reported previously^{89 (link)} according to a standardized nomenclature^{90 (link)}.

The laminar location determined by infrared videomicroscopy and recorded as 1–4 according to a location right within layers I, II/III, or IV. For neurons located at the border of layers I–II/III and II/III–IV, the laminar location was represented by 1.5 and 3.5, respectively. Somatic features were measured from IR DGC of the recorded neurons. Briefly, the soma was manually delineated using Image-Pro Analyzer 7.0 software (MediaCybernetics) and length of major and minor axes, perimeter and area were extracted. The soma elongation was calculated as the ratio between major and minor axis. Roundness was calculated according to: ${\displaystyle \frac{perimete{r}^2}{4\pi \times area}}$ ; a value close to one is indicative of round somata.