Five micron sections were prepared, deparaffinized, and then rehydrated. Each slide contained sections of well-oriented gastric glandular mucosa that extended along the greater curvature of the stomach. Slides were stained with hematoxylin and eosin for histology. Specific protein expression was identified by immunofluorescent staining. Antigen retrieval was performed using 10 mM citrate (pH 6). Slides were washed twice in phosphate-buffered saline containing 0.01% Triton X-100, incubated for 1 hour with the serum of the animal in which the secondary antibody was raised. Next, slides were incubated with the following antibodies overnight: Gastrin (DAKO, #A0568), Ki67 (Abcam, #15580), hIL1β (#10139-M201, Sino Biological, North Wales, PA), mIL1β (R&D, AF-401-NA), mIFN-γ (Interferon Source, #22100-3), Acetylated Tubulin (Sigma, #MABT868), and E-Cadherin (BD, #51-9001922). Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR; Invitrogen, Carlsbad, CA) were used to detect primary antibodies at a dilution of 1:500. Slides were mounted with Prolong gold anti-fade reagent with DAPI (Life Technologies, Rockville, MD). Immunofluorescence was visualized using the Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) or Olympus BX53F (Center Valley, PA).
Bx53f
Manufactured by Olympus
Sourced in Japan, China
The BX53F is a biological research microscope from Olympus. It features infinity-corrected optics and LED illumination. The microscope is designed for observation and documentation of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Dp72 microscope digital camera, by Olympus (3 mentions)
Szx16, by Olympus (3 mentions)
Hm340e, by Thermo Fisher Scientific (3 mentions)
C pace ep, by IonOptix (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Nicolet is50, by Thermo Fisher Scientific (3 mentions)
Antigen retrieval solution, by Agilent Technologies (2 mentions)
Cellsens, by Olympus (2 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (2 mentions)
Cellsens standard, by Olympus (2 mentions)
Fitc conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Pe conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Microtome, by Leica camera (2 mentions)
Fitc pna, by Merck Group (2 mentions)
Cellsens standard 1, by Olympus (2 mentions)
Hematoxylin solution, by Wuhan Servicebio Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Pentobarbital sodium, by Kyoritsu Seiyaku (2 mentions)
Lsm 880, by Zeiss (2 mentions)
166 protocols using bx53f
1
Histological and Immunofluorescent Analysis of Gastric Mucosa
2
Immunostaining of Cellular Markers
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Cells (5 × 104 cells/ml) cultured on glass coverslips in six-well plates were fixed with 4% paraformaldehyde and were incubated with the primary antibodies in PBS with 1% bovine serum albumin and 0.1% Triton X-100 at 4°C overnight. CD44 (Cell Signaling Technology, USA), β-catenin (Santa Cruz), Vimentin (Thermo Fisher Scientific), and TM4SF4 (Sigma-Aldrich) antibodies were used. Staining was visualized using an Alexa Fluor 488–conjugated anti-rabbit or Alexa Fluor 555–conjugated anti-mouse antibody (Invitrogen). Nuclei were counterstained using 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Stained cells were visualized with a fluorescence microscope (Olympus BX53F; Olympus, Tokyo, Japan).
3
TUNEL Assay for Apoptosis Detection
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The TUNEL assay was performed according to the manufacturer’s instructions (Promega Co., Madison, WI, USA). Briefly, after deparaffinization and rehydration, tissue sections were incubated with proteinase K (20 μg/mL) for 15 minutes at 24°C and washed in PBS. The slides were incubated with a mixture of TdT enzyme and biotinylated nucleotide for 60 minutes at 37°C. After being washed in PBS, the slides were incubated with streptavidin-horseradish peroxidase (HRP) solution for 30 minutes at 24°C. To detect TUNEL-positive signals, the slides were incubated with a mixture of 3,3’-diaminobenzidine (DAB) substrate, chromogen, and hydrogen peroxide. The slides were then examined and the images were recorded using a microscope (Olympus BX53F; Olympus, Tokyo, Japan). Dark brown staining represented a positive reaction. Positive reactions showed under the microscope, and stained cells were quantified by stereological analysis using Image-J software (https://imagej.nih.gov/ij/ ).
4
Fluorescence Microscopy of Fungal Hyphae
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According to previously reported methods [30 (link),31 (link)], G. saubinetii hyphae were cultured in potato dextrose broth (PDB) medium at 28 °C and 180 rpm for 24 h and then treated with 0, 2, and 4 eq. EC50 of compound E13 for 24 h. The PDB medium was removed by centrifuging at 4 °C and 6000 rpm for 5 min, and the hyphae were stained with 10 μL of propidium iodide (PI) solution (20 mg/L). The hyphae were incubated at 37 °C for 15 min in the dark and then washed with PBS three times. A coverslip was placed on the hyphae, and the samples were observed and photographed using an Olympus-BX53F fluorescence microscopy.
5
Histopathological Myocardial Injury Assessment
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Hematoxylin and eosin (H&E) staining detected histopathological injury to the myocardium. After the heart was isolated, the tissue was fixed, embedded, sliced, dewaxed, and stained. The sections were then observed under a light microscope (Olympus BX53F; Olympus Corp., Tokyo, Japan).
6
Cardiac and Intestinal Tissue Characterization
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After blood was taken, the thoracic cavity of the rats was completely exposed, the right atrial appendage was cut, and the abdominal aorta was immediately perfused with precooled phosphate-buffered saline (PBS). After perfusion, the heart and 2 cm of tissue in the middle region of the small intestine (ileum) were quickly removed and rinsed repeatedly with precooled 0.9% sodium chloride solution. The residual capsule and blood vessels were cut, dried with filter paper, and weighed with an electronic balance. The heart weight (HW) to body weight (BW) ratio was used as the heart weight index. Left ventricular myocardial tissue and ileum tissue were fixed with 10% neutral formaldehyde. Using conventional methods, specimens were dehydrated and embedded in paraffin, and sectioning was followed by hematoxylin and eosin (H&E) staining and Masson staining, which were analyzed using optical microscopy. The images were generated by using an optical microscope (model BX-53F, Olympus, Japan).
7
Microscopic Characterization of Pickering Emulsions
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Emulsion droplets were pipetted on microscope glass slides, after which optical microscope (Olympus BX53F, Tokyo, Japan) was used to observe and compare the shapes and microstructure of the ODF-SPI Pickering emulsions. The droplet size distributions were measured using the Image J program, counting at least 50 droplets from different images.
8
Immunohistochemical Staining of Microglia
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For IHC staining, brains were fixed in 10% phosphate-buffered formalin, routinely processed, and then embedded in paraffin. Brain sections were 4 µm thick sliced and placed onto glass slides. Before the staining protocol, slides were deparaffinized, rehydrated, and submerged in antigen retrieval solution (Dako, Jena, Germany) for 30 min at 100 °C. Non-specific binding was blocked with 3% peroxidase solution followed by blocking with Super Block (ScyTek Laboratories, Inc., Logan, UT, USA). The slides were incubated with rabbit anti-Iba-1(A19776, ABclonal) antibodies at 4 °C overnight. And then, were further incubated with horseradish peroxidase-conjugated secondary antibody (Vector Laboratories, Newark, CA, USA). Immune complexes were detected using DAB Substrate Kit (Vector Laboratories) according to the manufacturer’s instructions. Finally, tissues were counterstained and mounted. Images were analyzed under a light microscope (BX53F, Olympus Corp.) and digital imaging software (analySIS TS, Olympus Corp.).
9
Quantifying F. nucleatum Abundance via FISH
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Fluorescence in situ hybridization (FISH) was conducted to examine the abundance of F. nucleatum using a specific probe. Briefly, sections of formalin-fixed, paraffin-embedded colonic tissue were cut into 5 μm sections and hybridized in accordance with the manufacturer’s instructions (FOCOFISH, Guangzhou, China). The following universal bacterial probe (EUB338; Cy3-labeled) was used: 5’-GCTGCCTCCCGTAGGAGT-3’. The following probe specific to F. nucleatum (FUS664; FITC-labeled) was used: 5’-CTTGTAGTTCCGC(C/T)TACCTC-3’. The resulting slides were visualized and examined under a fluorescent microscope (BX53F; Olympus, Tokyo, Japan). Five random fields per sample (200 × magnification) were examined, and the average number of bacteria per field was calculated. A blinded reviewer examined five random fields per sample (200 × magnification), and the average number of bacteria per field was calculated. Demographics of patient tissues can be found in our previous study[23 (link)].
10
Histological Analysis of Mouse Liver
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After withdrawal of blood samples, mice were transcardially perfused with 40 mL of PBS and 50 mL of 10-% buffered neutral formalin (Thermo Scientific) as a fixative. After 24-h incubation of liver tissues in formalin, they were embedded in paraffin wax and cut into 5 μm thickness slices, followed by staining with haematoxilyn-eosin. Tissues have been photographed using an inverted Olympus BX53F (Olympus Co., Tokyo, Japan) equipped with a UPlanApo 20 × /NA 0.70 objective lens.
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