Zen 3.3 blue edition

For morphological diagnosis, sections (4 μm thick) were stained with hematoxylin and eosin (H&E). Masson's Tricromica Stain was used for the differential staining of collagen. All stained samples were examined under light and digital microscopes. The content of collagen fibers relative to the total adjacent normal tissue by image analysis using the software Zen 3.3 (blue edition, Zeiss) was also evaluated.

NET production over time and its effect on parasite entrapment/death were investigated by live cell imaging as previously described (32 (link)). Briefly, neutrophils (5 x 10⁵) were seeded onto 35 mm CELLview plates (Greiner Bio-One, Brazil) and allowed to adhere for 30 min at 37°C. Parasites were added (5:1 parasite/neutrophil ratio) in the presence of propidium iodide. NET release and parasite entrapment/death were monitored for 4.5 h on a Zeiss Axio Observer Z1 microscope equipped with Definitive Focus and a HMR Axiocam monochrome camera, with temperature stabilized at 37°C. Spontaneous death of parasites in NET-enriched supernatant or culture medium alone were also evaluated as control conditions. Image acquisition and analysis were performed using Zen 3.3 Blue Edition (Zeiss, Germany) and ImageJ 1.52 (National Institutes of Health, USA), respectively.

Immunofluorescence staining of the tissue sections was performed using ASMA (1:100; ab8211, Abcam, Cambridge, MA, USA, and images were captured using an Axioscan Z1 whole slide scanner (Zeiss, Jena, Germany). Pictures were evaluated using Zen 3.3 Blue Edition (Zeiss Microscopy, Jena, Germany). The fluorescence channel for the α-smooth muscle actin (αSMA) was dimmed to enhance the blood vessel signal, and the density of the αSMA per mm² was measured using the ImageJ software, version 1.53e (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), and Java 1.6.0_24 (64 bits). Counts matching 0.3 to 1.0 circularity were considered during our analysis.

The epicite^hydro samples taken from the wounds at the end of the in vivo experiments were stored in dry ice cooling until testing of the residual water content. Wet sample weight (mw) (n = 3) was determined directly after defrosting using an analytical balance (Kern ABJ 220-4NM). Afterwards, samples were air dried at room temperature until the mass achieved constancy. Dry mass (md) (n = 3) of samples was determined by weighing (Kern PCB 1000-2). The remaining water content (WC) of epicite^hydro samples was calculated using the following formula: WC = ((mw − md)/mw) * 100%. To confirm these results, primary dressing average thickness (in µm) was measured through H&E-stained slides in triplicate (each slide was measured 15 times, for a total of 45 measurements per treatment). BNC dressings were not removed before fixation and posterior staining, and manual measurements of the primary dressing thickness were obtained using the Zen 3.3 Blue Edition (Zeiss Microscopy, Jena, Germany) image processing software.