Cx21 light microscope

Hematological analysis was performed in all dogs as a part of their initial evaluation. Complete blood count was done using an Abacus hematology analyzer (Diatron, Budapest, Hungary). Blood smears were examined with a CX21 light microscope (Olympus, Tokyo, Japan) after May-Grünwald Giemsa staining.

Blood was collected from the abdominal vena cava under pentobarbital anesthesia, and the liver was removed. The blood was collected in EDTA-coated tubes and plasma was prepared by centrifugation. Alanine transaminase (ALT) and alkaline phosphatase (ALP) activities were measured in the plasma using commercial kits. For the histopathological evaluation, samples of the liver were fixed in formalin (10%). Paraffin-embedded sections of the liver were prepared and stained with haematoxylin and eosin before viewing with an Olympus CX21^® light microscope [65 (link)].

Thin and thick blood films were prepared for staining and parasite investigation. Blood films were sent to Tengchong Laboratory in a standard slide box for staining with 3% Giemsa and microscopic examination [18 ]. Parasitaemia was determined from thick blood films by counting the number of parasites per 200 WBCs. Thick blood films were classified as positive if one or more malaria parasites were observed and negative if no parasites were observed after examining at least 100 ‘oil-lens’ fields (i.e. at a magnification of ×1000). Thin smears were further examined after parasites were seen in the thick smears in order to measure parasitaemia and identify the species of malaria parasites. All the blood smears were examined using a CX21 light microscope (Olympus, Tokyo, Japan).
The malaria microscopist was blinded to BC-6800 results. Similarly, healthy controls were blinded to microscopy results. In order to assure malaria form quality of the microscopic examinations, the entire positive and 10% of the negative slides were sent to a senior malaria microscopist and re-examined at the Tengchong Laboratory. An experienced reader was assigned to re-examine the discrepant slides.

The hippocampi were cut into 400-μm-thick transverse sections with a tissue chopper (McIlwain, UK). These sections were postfixed in 2.5% glutaraldehyde for 1.5 h and 1% OsO₄ for 1 h (23 (link)). Samples were then dehydrated in an ascending series of ethanol followed by dry acetone and embedded in EPON resin (Sigma-Aldrich, Switzerland) per standard protocol (24 ). The semi-thin (1 μm) sections were produced with an LKB 8800 ultramicrotome (LKB, Sweden), stained with methylene blue, embedded in a Pertex mounting medium (HistoLab Products AB, Sweden), and visualized using an Olympus CX21 light microscope (Olympus Corp., Japan) (objective magnification−20x, NA−0.40). Ten to twelve semi-thin sections per animal and 2–3 images from each section were analyzed.
For electron microscopy, ultra-thin sections (60–70 nm) from the middle part of the CA1 stratum radiatum were stained with uranyl acetate and lead citrate and examined using a JEM-100CX transmission electron microscope (Jeol, Japan) at a magnification of ×10,000.

Tissue sections were stained by immunohistochemistry according to the XT-ultraView-DABv3 procedure. Tumor-infiltrating lymphocytes (TILs) and tumor-associated neutrophils (TANs) were assessed in tissue material stained with hematoxylin and eosin, and evaluated by a pathologist who was blinded to any patient clinical data, Fig. 1A. At the same time, the tissues were assessed for the presence of lymphoid follicles, Fig. 1C. To identify the types of lymphocytes, the following three antibodies were used: anti-CD3 (2GV6) rabbit monoclonal primary antibody (Ventana), anti-CD4 (SP35) rabbit monoclonal primary antibody (Ventana), anti-CD8 (SP57) rabbit monoclonal antibody (Ventana). Staining was performed automatically on the Benchmark XT using Ventana’s proprietary protocols. The histopathological assessment was performed using an Olympus CX21 light microscope at 400 × magnification. Tumor-infiltrating immune cells (TIICs) were detected in five randomly chosen, nonadjacent, nonoverlapping areas separately at the tumor center (TC) and at the invasive front (IF) to ensure uniformity and representativeness of the obtained results. Tumor-infiltrating immune cells were counted in 5 fields of view [FOV], the number of cells was calculated on 0.785 mm² of the tissue surface, and the result was the mean value [cells/FOV].

Thymus tissue was fixed in 10% formalin for 24 h followed by dehydration. The thymus was embedded in paraffin wax, sectioned into 5-µm slices and stained with Mayer’s hematoxylin and eosin (Merck Millipore, Darmstadt, Germany). Micrographs of the lung sections were captured with a CX21 light microscope (Olympus, Tokyo, Japan).

Purified protoplasts were counted using a blood cell count chamber under Olympus CX21 light microscope (Olympus, Japan). The yield was expressed as the number of protoplasts per gram fresh weight (g· FW). The viability was determined by fluorescein diacetate (FDA, Sigma-Aldrich, St. Louis, USA) staining according to Widholm [47 (link)]. The samples were incubated in dark for 5 min and then assessed under DMi8 inverted microscopy (Leica, Germany) with UV excitation light. Only viable protoplasts fluoresced bright green. The viability of the protoplasts was calculated by (viable protoplasts/total number of protoplasts) × 100%. For each sample, 3000 cells were analyzed in each replicate, and the counting was performed at least three times.