Small pieces of liver, kidney, and intestine were carefully collected, rinsed, and fixed in 10% neutral buffered formalin for at least 48 h, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin blocks. Sections were cut at 5 µm, dewaxed in xylene, and stained with Harris’s hematoxylin and eosin (H&E) to detect tissue degeneration and cell death, the Periodic acid Schiff(PAS) reaction to detect carbohydrate (mainly glycogen) content (Drury and Wallington 1980 ), or Masson’s trichrome method to detect fibrosis. Sections were visualized using an Olympus microscope (BX50F4, Olympus Optical Co., LTP, Japan).
Bx50f4
Manufactured by Olympus
Sourced in Japan
The BX50F4 is a microscope system designed for routine laboratory work. It features a four-lens turret for switching between magnification levels. The BX50F4 provides high-quality optical performance and is suitable for a variety of applications in research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Agilent Technologies (2 mentions)
Rm2135, by Leica camera (2 mentions)
Iba1 antibody, by Fujifilm (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Abc standard kit, by Vector Laboratories (1 mentions)
Cryostat, by Leica Biosystems (1 mentions)
Tu 213, by Yamato Scientific (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Api 20ne, by bioMérieux (1 mentions)
Dako pen, by Agilent Technologies (1 mentions)
Hematoxylin 2, by Roche (1 mentions)
Shandon varistain v24 4, by Thermo Fisher Scientific (1 mentions)
Ab155282, by Abcam (1 mentions)
Dc platten kieselgel 60 f254, by Merck Group (1 mentions)
Cryostat, by Thermo Fisher Scientific (1 mentions)
Pkh26 red fluorescent cell linker kit, by Merck Group (1 mentions)
Anti cd31, by Agilent Technologies (1 mentions)
Pkh26, by Merck Group (1 mentions)
34 protocols using bx50f4
1
Histological Analysis of Organ Tissues
2
Histological Analysis of Liver and Kidney
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The liver and kidney samples were taken and then fixed in 10% neutral buffered formalin. Fixed samples were processed routinely using a paraffin embedding technique, then sectioned at 5 μm in thickness and stained with Harris’ hematoxylin and eosin (H&E). Sections were examined using an Olympus microscope model BX50F4 (Olympus Optical Co., Ltd., Tokyo, Japan).
3
Histopathological Examination of Thyroid and Gonad
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For thyroid follicle examination, the fixed fish heads were placed in 10% EDTA (PH 7.4) solution for 1 week for decalcification. Then, fixed gonad and EDTA-treated head samples were dehydrated through arising grades of ethanol and then were cleared and embedded in paraffin wax. For thyroid follicle sections, serial frontal sections were cut at 7 μm from the ventral side of the head. The embedded gonad samples were sectioned at 5 µm thickness; the samples were stained with H&E stain and then observed microscopically (Hamed et al., 2021 (link); Sayed E.-D. H. et al., 2022 (link); Sayed et al., 2023b (link)). For scoring each histopathological parameter, six sections of four fish from each treatment were randomly selected and labeled as follows: control, unchanged (0–2); mild, + (>2–10%) area of section; moderate, ++ (>10–40%) partition area; and severe, +++ (>40% partition area) (Hamed et al., 2021 (link)). The sections were examined under an Olympus microscope (model BX50F4, Olympus Optical Co., Ltd., Tokyo, Japan).
4
Immunohistochemical Staining of Iba1+ Microglia
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Under deep anesthesia with the M/M/B anesthetic mixture, mice were perfused through the ascending aorta with 0.9% NaCl followed by 4% paraformaldehyde/0.1 M phosphate buffer (pH 7.4). The brains were carefully dissected out of the skull and immersed in 10% to 30% sucrose solution for cryoprotection. Coronal sections of 50 μm thickness were prepared using a cryostat (Leica Biosystems, Wetzlar, Germany). The sections were treated with 0.3% Triton X followed by 3% H 2 O 2 for 10 min. After rinsing in phosphate buffered saline (PBS), the sections were incubated with 10% bovine serum albumin (Thermo Fisher Science, Waltham, MA, USA) for 1 h, followed by incubation with rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1) antibody (1:1000, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 4 °C overnight. After rinsing with PBS, sections were treated with biotinylated goat anti-rabbit IgG (1:1000, Vector Laboratories, Burlingame, CA, USA) for 2 h, rinsed in PBS, and transferred to an ABC Standard kit (Vector Laboratories) for 2 h. The slides were rinsed in PBS again and stained with 3,3'-diaminobenzidine tetrahydrochloride (Dojin Glocal Co. Ltd., Kumamoto, Japan). The slides were observed under a light microscope (BX50F4; Olympus Optical Co., Tokyo, Japan), and the images were captured using a chargedcoupled device (CCD) camera (DP71, Olympus Optical Co.).
5
Radial Culm Wall Anatomy
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Transverse sections of 20 μm in thickness, including the whole radial culm wall, were cut with a sliding microtome (Yamato Kohki TU-213, Saitama, Japan). All sections were observed directly under light microscope (Olympus BX50F4, Tokyo, Japan) for checking integrity of vascular bundles. Three sections from each sample block as repetition were used for detection of autofluorescence of cell walls by fluorescence microscopy before and after chemical treatments. The average value of nine sections from three sample blocks was as the value of lignin content.
6
Histological Analysis of Brain Regions
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Histological changes were evaluated in the SVZ (roof, DL-SVZ, and L-SVZ), caudate nucleus, and periventricular white matter brain regions at both bregma −2.0 and −4.0 levels (Supplementary Figures 1 , 2 ) by using H&E staining and analyzed with Fiji/ImageJ image software. For each animal, level, and section, a total of fifteen non-overlapping microphotographs (3 from each area) were taken at 400 × magnification in a light field optical microscope (Olympus BX50F4, Japan). A blinded histologist counted morphologically well-preserved cells (undamaged), together with cells with apoptotic or necrotic features. Apoptotic-like cells were characterized by the presence of nuclear karyorrhexis and low cytoplasmic change, whereas necrotic cells were identified by a pyknotic nucleus or no nucleus, along with a swollen, eosinophilic cytoplasm (17 (link)). We did not count apoptotic nor necrotic profiles that were within or adjacent to blood vessels to avoid including apoptotic/necrotic endothelial and white blood cells. The undamaged, apoptotic and necrotic cell count was averaged from 3 high-power fields from 3 slides from the same region in each animal, and values are given as cells per mm2.
7
Phenotypic and Chemotaxonomic Characterization of Achromobacter xylosoxidans H1_3_1
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Achromobacter xylosoxidans strain H1_3_1 was grown on nutrient agar (Oxoid) at 30°C under aerobic conditions for 2 days and observed using a light microscope (Olympus BX50F4). Conventional phenotypic tests were performed as described in Barrow and Feltham (1993) . The activity of constitutive enzymes and other physiological properties were determined using the API 20NE and API ZYM microtest systems (bioMérieux) according to the manufacturer’s instructions. For cell fatty acid analysis, strain H1_3_1 was cultivated on TSBA (Becton Dickinson) at 30°C under aerobic conditions for 24 h. The cells were freeze-dried, and fatty acid methyl esters were extracted and analyzed using the Sherlock Microbial Identification System (ver.6.0) according to the manufacturer’s procedures. Analysis of respiratory quinones was carried out by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany). Respiratory quinones are extracted from freeze-dried cell material using hexane, purified by a silica-based solid phase extraction, and analyzed by HPLC recording absorption spectra (Vieira et al., 2021 (link)). For relative quantification, 270 nm for ubiquinones and 326 nm for menaquinones are used.
8
Histological Analysis of Mouse Lung Tissue
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Mice were anesthetized by intraperitoneal injection of 10 % pentobarbital (100 mg/kg body weight), and were sacrificed for collection of blood and excision of organs. The lungs were inflated intratracheally under constant positive pressure at 25 cm H2O with 4 % paraformaldehyde for fixation and resected to prepare paraffin-embedded lung blocks for morphological examinations (Hirama et al. 2007 (link)). Other tissues were also fixed with 4 % paraformaldehyde, and embedded into paraffin. Tissue sections were cut at a 3-μm thickness and placed on glass slides, subsequently stained with hematoxylin and eosin, and examined using a microscope (BX50F4; Olympus, Tokyo, Japan).
9
Comprehensive Urinalysis Analysis
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Urinalysis was assessed on day 0 and day 90 before sacrifice. Urine volume, specific gravity, bilirubin, pH, protein, glucose, ketones, nitrite, urobilinogen, and occult blood (Oc. blood) were tested by a Clinitex 100 Urine Chemistry Analyzer (Miles Inc. Diagnostic Division, Elkhart, IN, USA). Urinary sediments of RBC, leukocytes, epithelial cells (epithelia), casts, and crystals were observed by optical microscope (Olympus BX50F4, Tokyo, Japan).
10
Immunofluorescence Tissue Staining Protocol
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Our method was similar to Geetika Singh's [13 (link)] (2016) (p.463), as shown in Table 2 . A rotatory microtome was used to cut FFPE tissue blocks at 3 μ (Leica RM2135; Nussloch, Germany). Deparaffinization of slides was performed for 3 minutes each with two changes of xylene, and then rehydrated with 100 percent alcohol twice for 3 minutes each, 95 percent alcohol for 1 minute, and, eventually, 70 percent alcohol for 60 seconds. The next step was rinsing slides for 3 min and using a Dako pen to mark tissues. Slides were triplet-washed using PBS, each lasting 10 min. The next step was the incubation of slides using proteinase K (ready to use, code no. S3020; Dako, CA, USA) for 60 minutes. Then, slides were rinsed in triplets using PBS, which each lasted for 10 min. The next step was the incubation of slides using a primary antibody. Afterward, slides were rinsed using PBS three times; each lasted for 10 min. The last stage was mounting slides using glycerol and examination under an immunofluorescence microscope (BX50F4; Olympus).
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