Binocular light microscope

Serial sections (8 semi-serial sections of each maxilla, with a 5 μm thickness for each section) were obtained using a microtome (Leica RM2255, Germany) and stained with HE (hematoxylin and eosin). Morphometric measurements were performed by a single calibrated investigator with a binocular light microscope (Olympus Optical Co, Tokyo, Japan) using a 100× immersion objective and a Zeiss kpl 8× eyepiece containing a Zeiss II integration grid (Carl Zeiss Jena GmbH, Jena, Germany) with 10 parallel lines and 100 points in a quadrangular area. The grid image was successively superimposed on approximately 13 histological fields per histological section, comprised of all tooth sockets from the coronal limit adjacent to the gingival epithelium until the lower apical limit. For each animal/socket, sections from the medial were evaluated. In the morphometric analysis, points were counted coinciding with the images of the following components of the alveolar socket: clot, inflammatory cells, blood vessels, fibroblasts, collagen fibers, bone matrix, osteoblasts, osteoclasts and other components (empty space left by extracellular liquid and bone marrow); similar to previous descriptions (25 (link), 47 (link)–50 (link)). The results were presented as the mean of volume density for each evaluated structure.

After μCT scanning, maxillae were immersed in buffered 4% EDTA for demineralization and processing for embedding in paraffin blocks. Transversal serial 5-µm slices from medial third were cut for histology with H&E staining, picrosirius red, immunohistochemistry, and immunofluorescence. A total of three histological sections from central region of the alveolar socket stained by H&E were used to quantify the following healing components: clot formation, inflammatory infiltrate, connective tissue (collagen fibers, fibroblasts, and blood vessels), bone matrix, osteoblasts, osteoclasts, and other components (empty spaces and bone marrow), as previously described (7 (link)). The identification and quantification of healing components was performed by a single calibrated investigator with a binocular light microscope (Olympus Optical Co., Tokyo, Japan) using a 100× immersion objective and a Zeiss kpl 8× eyepiece containing a Zeiss II integration grid (Carl Zeiss Jena GmbH, Jena, Germany) with 100 points in a quadrangular area. The grid image was successively superimposed on 13 histological regions per histological section, totaling 3 sections for each specimen. Only the points coincident with the histological components were considered, and the total number of points was obtained to calculate the area density for each healing component in each section.

The analysis of immunolabeled cells was performed by a single calibrated investigator with a binocular light microscope (Olympus Optical Co., Tokyo, Japan) using a 100× immersion objective, following the similar criteria described previously for histomorphometric analysis in H&E (see Histology Sample Preparation and Histomorphometric Analysis). Briefly, five samples (biological replicate) for each experimental period and strains were used for quantitative analysis. A total of three sections of each sample (technical replicate) containing the central region of the alveolar socket was used to quantify immunolabeled cells for each mentioned target (F4/80, CCR2, CCR5, Ly6g-Gr1, CD3, and CD68). A total of 13 fields (100 points in a quadrangular area) were analyzed using Zeiss II integration grid (100 points) (Carl Zeiss Jena GmbH, Jena, Germany) for each section. Only the points coincident with the immunolabeled cells were considered in cell counting, and the mean for each section was obtained for statistical analysis.

For the MN evaluation, from each slide, 400 cells were scored for computing the MNF. A binocular light microscope (Olympus, Japan) was used to examine the micronucleated cells (100×). The method described by Tolbert et al. [28 (link)] was used to score the micronucleus frequency.

Totally 12 malaria blood film slide panels, 6 Giemsa-stained and 6 unstained slides, were administered for assessment of performance in parasites detection, species identification, and quantification. The standardized malaria blood film slide panels were distributed in the following approach: Two negative stained and unstained slides; 2 P. falciparum stained and unstained slides of low density (140/μl); 2 P. falciparum stained and unstained slides of high density (79,640/μl); 2 P. vivax stained and unstained slides of low density (440/μl); 2 P. vivax stained and unstained slides of high density (54,400/μl); 2 mixed species (Pf + Pv) of stained and unstained blood film slides. Blood films were stained with similar Giemsa solution (prepared from similar stock solution and buffer from the central laboratory) by each malaria microscopist under evaluation in all facilities. All study participants used Olympus binocular light microscope equipped with eyepieces (X10) and objectives (x10, x40 and x100), and the same brand immersion oil was used during microscopic examination. Based on WHO recommendation, quantification result of participant was considered correct when it was in between 25% ± the mean calculated from result of expert readers. A total of 120 minutes (10 minutes per each slide) was allocated to examine the 12 malaria slides [4 (link)].