Bx61 light microscope

Midribs from the fifth leaf and stem tissue taken from ~2 cm from the bottom of Arabidopsis plant were collected from 40-day-old PeUGDH4 transgenic and wild-type Arabidopsis plants. These tissues were fixed in the solution including 75% ethanol and 25% acetic acid. The procedures of paraffin sectioning as described by Cai and Lashbrook^{57 (link)}. After that, sections were dewaxing for 30 min and stained for 2–3 min in 0.02% toluidine blue (Sigma, USA), imaged using an Olympus BX-61 light microscope (Olympus Co.).

Animals were sacrificed by anaesthesia overdose and transcardially perfused with 1x PBS for the analysis of lung injury. The lungs were dissected, and one-third of each lobe for histological and immunofluorescence analysis was fixed in 4% paraformaldehyde (PFA) for 24 h at room temperature (RT), embedded in paraffin, and cut into 3–5 μm-thick-sections. The sections used for histological studies were stained with H&E, and two researchers independently evaluated the slides in a double-blind manner. About 20% of the total lung tissue per section of each animal was photographed in the Olympus BX61 light microscope (Olympus BX61 Upright Microscope, Tokyo, Japan) coupled with Olympus DP70 digital camera using a magnification of 400×. The lung injury score was quantified considering alveolar wall thickness, congestion of the alveoli, immune cell infiltration, number of macrophages and neutrophils, and formation of hyaline membranes. The semiquantitative score, ranging from zero to twenty-three, was independently assessed by two different individuals in a blinded manner (Table 1). Two independent experiments were conducted and subjected to statistical analysis. Since both experiments yielded statistical differences, the data from one of them are presented herein.

For evaluation of endocan, the immunoreactive score (IRS) was obtained by multiplying the staining intensity (SI: 0 = negative; 1 = weak; 2 = intermediate; 3 = strong) and percentage of positive tumor/endothelial cells (PPC: 0 = 0 %; 1 = 1–10 %; 2 = 11–50 %, 3 = 51–80 %; 4 = >80 %) as established previously 24. IRS = SI × PPC. Five high power fields (HPFs) (400×) were randomly selected from each slide for IRS calculation. To determine the MVD, tissue sections stained with CD105 and CD34 were examined using an Olympus BX61 light microscope. According to the methods exposed by Weidner previously [25 (link)]. The sections were firstly examined at 100× magnification for the location rich of blood vessels. Then, pictures of five different fields were taken at 200× magnification. Finally, the number of blood vessels in each picture was counted independently by two researchers. Every single cell and cell cluster stained were assessed as a blood vessel, regardless of whether the structure of vascular lumen was observed. Every 40 microns of one large lumen was recorded as one blood vessel. The average value of five fields was recorded as the MVD score [25 (link)].

Fore and hind legs were removed by forceps from F1 of UAS-OctαR-dsRNAi × mhc-GAL4. Then transferred to glass slides using a fine brush for microscopic examination using an Olympus BX61 light microscope at magnification 2.5 × 10. Photographs were taken and the length and width of each leg segment (coxa, trochanter, femur, tibia and tarsi) were measured using ImageJ software. In parallel, F1 of w¹¹¹⁸ crossed to: (i) OctαR-dsRNAi and (ii) GAL4-mhc was used as controls for RNAi experiment. Additionally, morphometric analysis of OctαR KO adult legs was done. W¹¹¹⁸ was used as control for knock out (KO) experiment.

The fixed brain and liver samples were dehydrated using increasing ethanol grades, cleared in xylene, and finally embedded in paraffin; subsequently, paraffin sections were obtained (4 μm thickness). These sections were stained with hematoxylin and eosin (H&E) as reported previously by Suvarna et al. [29 ]. The slides were examined using an Olympus BX61 light microscope (Tokyo, Japan).
For lesion scoring, five non-repeated, randomly selected microscopic fields (magnification, 40×) were examined in three different slides per animal/group. The mean values of the five microscopic fields examined were considered the final lesion score of each animal. The histopathological lesions observed in the liver, cerebellar, and cerebral cortices, and hippocampus in all examined groups were assessed using the following scoring method; 0, _1, _2, _3, _4, and _5 corresponding to no change, mild change, mild-to-moderate change, moderate change, moderate-to-severe change, and severe change, respectively [30 (link)].

Common starlings were caught using permanent traps (large ‘Rybachy’ type, zigzag and funnel traps) and mist nets at the Ventes Ragas Ornithological station (55°20′38.93” N, 21°11′34.05” E), Lithuania in May 2019. This period corresponded to the beginning of the breeding season of Common starlings at the study site. Blood samples were collected by puncturing the branchial vein and fixed in SET-buffer [28 (link)] for further molecular analyses. A drop of fresh blood was used to prepare 2–13 blood films on ready-to-use glass slides. The films were fixed by immersion of the slides in absolute methanol for one second and then stained with 10% Giemsa [1 ]. In all, 19 Common starlings were sampled. Microscopic examination of blood films determined the presence of haemosporidians, and species of H. pastoris was identified [1 ]. Later, the species identification was confirmed by using molecular barcoding in the laboratory (see description below). Microscopic examination of blood films was performed using a BX61 light microscope (Olympus, Tokyo, Japan). Five microscopy-positive birds with gametocyte parasitemia between 1 and 26% of infected red blood cells (calculated according to [29 (link)]) were euthanized (by decapitation) and their organs were processed for histological examination.