Bx53 light microscope

Invasion assays were performed using BD Biocoat Matrigel Invasion Chambers with 8-μm pore filters (BD Biosciences). The bottom wells of the companion plate were filled with 20% serum-containing medium to act as a chemoattractant for the cells. Cells were seeded at a density of 2.5 x 10⁴ per insert in serum-free medium. Invasion assays were incubated for approximately 16 hours at 37°C, 5% CO₂, stained with a Diff-Quick stain set (Fisher Scientific) and imaged with an Olympus BX53 light microscope (Olympus America). Invasive cells were counted in five representative high-power fields and all experiments were performed three times.
For invasion experiments involving cells treated with inhibitors, cells were pretreated with the respective dose of inhibitor two hours prior to being harvested for the assay. Inhibitors and vehicle controls were also added to the serum-free medium cell suspension prior to addition into the invasion chambers.

mSMG tissues were immersed in 10% formalin at room temperature for one day, dehydrated in serial ethanol solutions (50%, 70% and 100% for 2 h each), embedded in paraffin wax and cut into 7 μm sections. mSMG sections from each group were deparaffinized with xylene and rehydrated with serial ethanol solutions (100%, 70% and 50%) and distilled water. Then, hematoxylin and eosin (H&E) and picrosirius red staining were performed, and tissue sections were subjected to a blind histopathological analysis using a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany) as well as an Olympus BX53 Light Microscope (Olympus America, Center Valley, PA). In addition, the ratio of acinar structures to ductal structures was analyzed using ImageJ and GraphPad Prism 6.

To compare the microscopic structure of internodes in WT and mutant plants, the eighth internodes of the stems of 40-day-old plants of TopMark and M406 were fixed, rinsed, postfixed, washed, dehydrated, and embedded. Semithin sections were prepared with Formvar-coated gold grids and observed with an Olympus-BX53 light microscope as previously described^{46 (link)}.

Collected specimens were immediately subjected to a rapid cooling shock at 2°C–4°C by submersion in water mixed with ice at a ratio of 1:1 (Wilson et al. 2009 (link)). All fish were measured for their total length (TL) and total weight (TW). Blood samples of 0.3 mL–0.5 mL were taken from 10 fish per species/region, drawn from the heart area with a 1 mL plastic syringe, needle No. 21G. Following the standard method of Singkhanan et al. (2019) , approximately 10 μL of each sample were smeared on a glass slide and left to dry at room temperature, forming a thin film. The prepared slides were treated with methyl alcohol for 1 min, stained with Wright’s Giemsa for 15–25 min, and the dye was then washed off with a pH 7.4 buffer solution for 30 min. The morphology of blood cells on the slides was then studied (Cossins & Gibson 1997 (link)). The blood cell typing and morphological analysis including erythrocyte length (EW), erythrocyte width (EL), nucleus length (NL) and nucleus width (NW), blood cell percentages, and blood cell abnormalities were evaluated according to the standards of Andreyeva and Mukhanov (2012) (link), which were performed using Olympus BX53 light microscope (Olympus Corporation, Tokyo, Japan). Total abnormal erythrocytes (from 100 cells/fish per slide) and leucocytes (from 10 cells/fish per slide) were counted, and then calculated as percentages.

For histological sections, three individuals without visible lesions were selected from each experimental group. Fish were immortalized in MS-222 solution (10 mg L⁻¹), after which they were fixed in Davidson’s solution for 24 h. Then, tissue samples were dehydrated in a series of graded alcohols and embedded in paraffin. To obtain sections of the anterior/middle and posterior intestines of each fish, serial slices (4 μm) in the frontal plane were made and stained with hematoxylin and eosin (H&E). Histological sections were prepared and stained according to Suvarna et al. [66 ]. In addition, morphometric measurements of the intestines of three specimens of D. rerio (50 measurements per section) were performed according to the following parameters: height of the adsorbing epithelium, width of lamina propria, goblet cells area, number of goblet cells per 100 µm of epithelium, and thickness of the muscularis.
Histological preparations were examined under an Olympus BX53 light microscope (Olympus Corporation, Tokyo, Japan) with CarlZeiss ERc 5 s (Zeiss, Oberkochen, Germany) and ToupCam 16.0 MP (ToupTek Photonics, Hangzhou, China) ocular attachments using ZEN lite software (Zeiss, Oberkochen, Germany) and ToupCam view 16.0 (ToupTek Photonics, Hangzhou, China).