Qwin software

Agar diffusion method was selected to verify the antibacterial effectiveness of silver-enriched collagen and alginate beads. Antibacterial activity was tested against the Gram-positive Staphylococcus epidermidis (ATCC 12228) and the Gram-negative Pseudomonas aeruginosa (ATCC 10145), based on The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [16 (link)], with some modifications. Briefly, Mueller-Hinton agar was used as the bacterial growth medium. The agar surface was inoculated by using a swab dipped in a cell suspension of a 0.5 McFarland turbidity standard, approximately corresponding to 1 × 10⁸ to 2 × 10⁸ colony forming unit per ml (CFU/mL). Silver-enriched beads were placed on each S. epidermidis and P. aeruginosa inoculated agar plate by means of sterile tweezers and incubated at 37 °C for 20 h. The positive control consisted of collagen and alginate beads enriched with PenStrep 20× (Sigma-Aldrich (Milan, Italy)), negative controls were made of collagen and alginate scaffolds only. Afterwards, the inhibition zone areas were measured by QWin software (Leica) after digital acquisitions. The experiments were carried out in triplicate in four experimental replicates for both bacterial strains with regard to alginate (n = 12) and in triplicate in two experimental replicates with regard to collagen (n = 6).

Femurs were collected and fixed in 10% buffered formalin for 24 h at 4 °C and were decalcified with 10% neutralized EDTA (Sigma) for 4 weeks; then dehydrated with an ascending series of ethanol concentrations, cleared in xylene and embedded in Paraplast (Sigma). Histological sections (6 µm) were stained with hematoxylin-eosin (H&E) for general morphologic analysis or picrosirius for collagen fibers (type I and type III) quantification and stereological analysis [50 (link)]. The color displayed under polarizing microscopy was a result of fiber thickness, as well as the arrangement and packing of the collagen molecules. Normal tightly packed thick collagen fibers had polarization colors in the red spectrum while thin or unpacked fibers had green birefringence [51 (link)]. Sections were observed under normal and polarized light, and digitalized images were analyzed using Leica Q-win software (Version 3.0) to calculate mean collagen fiber area.
Non-injured bone was used to show differences with our injured groups in radiographic evaluation and histological analysis (H&E and picrosirius).

After animal sacrifice, tissue samples were embedded in OCT tissue-freezing medium, frozen in liquid nitrogen, and stored at −80°C until analysis. Samples were fixed with 10% animal buffered formalin for a minimum period of 48 h. Tissues were incorporated into the paraffin, sections were cut to 5–10 μm and stained with standard techniques with hematoxylin and eosin (H.E.), Mallory trichrome stain, and periodic acid-Schiff (PAS). Other specimens stored at −80 were used for ATPase and Succinodehydrogenase stains in cryo-section. Muscles were cross-sectioned using a microtome. Digital images were taken from the cross-section at 10-100x magnification to evaluate muscle fiber morphology and to determine fiber cross-sectional area (CSA) measures. Images from 20 random fields were acquired for the ten stained sections of each specimen using a D 4000 Leica DMLS microscope equipped with a camera and image analyzer NIS elementes-BR-Nikon. Section analysis and the CSA evaluation of the fibers was performed by QWin software (Leica). Of the subjects sacrificed, a gross necropsy of all organs was also carried out (Trapani et al., 2017 (link)). Examination of cellular morphology as well as of intracellular structures was conducted and the severity of the observed lesions was assessed (Gibson-Corley et al., 2013 (link)), by animal pathologists blinded to mouse genotype.

Live animals, WISH samples mounted with Mowiol mounting medium, were photographed using a microscope (SteREODiscovery.V20; Carl Zeiss, Jena, Germany) equipped with a Plan Apochromat ×1.0 objective and a digital microscope camera (AxioCamHRc; Carl Zeiss) automated using the AxioVision Rel.4.8 software (Carl Zeiss). FISH specimens were mounted with fluorescence mounting medium (Dako, Glostrup, Denmark) or Mowiol mounting medium, and the images were captured with a laser-scanning confocal microscope (True Confocal Scanner SP5; Leica; HCX Plan Apochromat confocal scanning ×10/0.4 NA, ×20/0.7 NA, ×40/0.85 NA or ×63/1.40 NA oil immersion objective lens) using the LAS AF software (Leica). Images were processed with the LAS AF Lite software and Photoshop software (Adobe, San Jose, CA, USA) and were quantified using the QWin software (Leica) or the ImageJ software (National Institutes of Health, Bethesda, MD, USA). All ISH experiments were performed, imaged and processed identically (at room temperature, 22 °C) to allow direct comparison between experimental animals and controls.

Establishment of breast precancerous lesion rat model and the animal experiments were performed after the approval from the Laboratory Animal Ethics Committee of Jinan University. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and related ethical regulations of Jinan University.
After the animal experiments were terminated at the end of the 14th week, the paraffin sample of rat breast tissues in different experimental groups were serially sectioned (4 μm), conventionally baked, dewaxed and hydrated. The routine immunohistochemical streptavidin-peroxidase (SP) procedures were conducted [42 (link)]. The protein expression was observed under light microscope. The positive cells expression average area percentage was analyzed using Leica Qwin software. The statistical analysis was conducted.