Dp12 digital camera

For immunohistochemical staining, spinal cord sections were subjected to antigen retrieval using a microwave in 0.01 mol/L citrate solution for 15 min, then incubated with rabbit anti-CD3, anti-CD4 and anti-CD8 antibodies (Abcam kit). The sections were then incubated with a streptavidin-biotin-peroxidase complex (SABC) kit for 30 min and stained with 3,3′-diaminobenzidine (DAB). The mounted sections were sealed with epoxy resin, and the results were observed under a light microscope (Olympus BX51 microscope with an Olympus DP12 digital camera).

Tube formation assay was performed as elsewhere described [29 (link), 30 (link)]. Briefly, 48 h following lentiviral transfection, HUVECs (2 × 10⁴ per well) were seeded in duplicate onto 96-well culture dishes coated with 50 μl of Matrigel (BD Biosciences). Tube formation was observed every 3 h post seeding with an inverted Olympus phase-contrast microscope, and five high-power fields at 100 magnifications were imaged randomly by using Olympus DP12 digital camera. The vessel length and branch points for each group were quantified by Angiogenesis Analyzer, a plug-in of ImageJ software. This experiment was independently repeated three times.

For histological assessment, brains were fixed in 10% paraformaldehyde solution. The degree of demyelination in the corpus callosum was assessed by Luxol Fast Blue (LFB) staining (Sigma, St. Louis, MO, USA). Briefly, paraffin-embedded samples were put on slides, deparaffinized, rehydrated by reducing concentrations of ethanol and placed in the LFB solution (0.01%) at 60 °C. To distinguish between white and gray matter, tissues were differentiated using a lithium carbonate solution (0.05%, Merck, Darmstadt, Germany). The stained tissues were observed and captured using a light microscope (Olympus BX51 microscope with an Olympus DP12 digital camera). The clear-white areas of demyelination relative to the blue normal tissue were measured and quantified using a total of 10 random sections per mouse.

The bone cores obtained were individually preserved in 10% formaldehyde for 20 days. The samples were then decalcified for 30 days using TBD-2 (Anatomical Pathology International, Runcorn, Cheshire, UK). After dehydration and inclusion in paraffin, sections of 20 μm thickness were prepared; samples were stained with picrosirius-hematoxylin and hematoxylin to distinguish between immature and mature bone.
For histomorphometric analysis, images were enlarged 20×, and eight fields per sample were evaluated digitally (using a DP12 digital camera, Olympus, Nagano, Japan). Microimage 4.0 software (Media Cybernetics, Silver Spring, MD, USA) was used for image analysis.
All analyses were carried out by the same technician, who was blinded to each sample’s group assignation (test or control).
The total area of newly formed bone and connective tissue were evaluated and the percentage of immature bone was measured.

Blood films were screened using a BX41 (Olympus, Tokyo, Japan) light microscope to determine the infection status of the collected birds and the prevalence of the parasite (percentage of birds infected with the target parasites species out of all collected individuals of the same host species). Blood films were analyzed for 15–20 min at medium magnification (400×) followed by examination of 100 microscope fields at high magnification (1000×). For dissected individuals with available blood films, the intensity of parasitaemia was determined by counting the number of parasites per 1000 erythrocytes or per 10,000 erythrocytes in case of low parasitaemia (Godfrey et al., 1987 (link)); measurements and images of gametocytes were taken using an Olympus DP12 digital camera and the Olympus DP-SOFT software. The standard range of Haemoproteus parasite characters were used for morphological characterization of gametocytes and their host cells for a new Haemoproteus sp. (Valkiūnas, 2005 ).

An Olympus BX51 light microscope (Olympus) equipped with an Olympus DP12 digital camera and Olympus DP-SOFT imaging software was used to examine H&E stained histological sections. First, each histological preparation was examined at medium magnification (400×). If present, tissue stages of haemosporidian parasites can be readily visible. If exoerythrocytic meronts were found, they were examined and illustrated under set of different magnifications (100, 200, 400 and 1000×) for better visualization of parasite location and structure.

Infected birds were determined using PCR-based testing of blood samples, and blood films of the PCR-positive samples were examined under microscope. In order to perform morphological analysis of Lankesterella parasites, blood films containing different lineages were selected based on DNA sequence information; these preparations were carefully examined at 1000× magnification (oil immersion) using an Olympus BX41 light microscope equipped with an Olympus DP-12 digital camera and the Olympus DP-Soft v. 3.2 imaging software (Olympus, Tokyo, Japan). The intensity of parasitemia was determined by counting the number of parasites per 100 thrombocytes or leukocytes. Illustrations and measurements were prepared using the same instruments. Representative blood films were deposited in the Nature Research Centre, Vilnius, Lithuania and in the Queensland Museum, Brisbane, Australia (see parasite descriptions for accession numbers of these preparations).