Sexual structures of the collected specimens were used for morphological observations and identification. The stroma and perithecia were observed, photographed and measured with a VHX-600E 3D microscope from the Keyence Corporation (Osaka, Japan). Fresh material was respectively immersed in water, 10% KOH, and Melzer’s reagent to observe micromorphological structures as determined by Ma et al. and Song et al. [20 (link),21 (link)]. The observations, micrographs, and measurements of asci and ascospores were performed by using an Olympus IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan) and the CellSens Dimensions Software (Olympus, Tokyo, Japan). The observations and photographs of ornamentation of ascospores were examined by scanning electron microscope (SEM) (Phenom Corporation, Netherlands) as given in Friebes and Wendelin [23 ]. The stromatal color and KOH-extractable pigments were assigned following the mycological color chart of Rayner [24 ]. The present paper contains the following abbreviations: KOH = 10% potassium hydroxide; n = number of measuring objects; M = arithmetical average of sizes of all measuring objects.
Ix73 inverted fluorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The IX73 is an inverted fluorescence microscope designed for advanced microscopy applications. It features a high-performance optical system and is capable of various imaging techniques, including brightfield, phase contrast, and fluorescence microscopy.
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Lab products found in correlation
Ab6717, by Abcam (3 mentions)
Live dead assay, by Thermo Fisher Scientific (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Cellsens dimension imaging software, by Olympus (2 mentions)
Bs 9353r, by Bioss Antibodies (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Cellsens dimensions software, by Olympus (1 mentions)
Cm1950 frozen slicer, by Leica (1 mentions)
Ab288751, by Abcam (1 mentions)
Ab280209, by Abcam (1 mentions)
Sc 8404, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Nucview 488, by Biotium (1 mentions)
Apc 30dr, by Astec (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Homocysteine, by Merck Group (1 mentions)
Isoleucine, by Merck Group (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Acetaminophen, by Merck Group (1 mentions)
58 protocols using ix73 inverted fluorescence microscope
1
Morphological Characterization of Fungal Specimens
2
Immunofluorescence Analysis of Kidney Tissues
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Other portions of the fresh tissues were immediately embedded in tissue freezing media optimum cutting temperature (OCT) compound. The kidney samples were then frozen at À20 C, and cut into 5-lm slices using a CM1950 Frozen Slicer (Leica, Nussloch, Germany). The sections were then fixed in pre-cooled acetone for 5 min, permeabilized using 0.1% Triton X-100 (in PBS) for 15 min, and then blocked for 2 h at RT with 5% fetal bovine serum in PBS. For double-labeling, the sections were coincubated at 4 C overnight with rabbit anti-CK18 antibody (1:100 dilution in PBS) and goat anti-B1R/B2R antibody (1:100 in PBS). On the following day, the sections were warmed to RT, rinsed three times in PBS, then incubated for 2 h at RT in PBS containing fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, i.e. a mixture of donkey anti-rabbit (red) IgG-FITC (1:200 in PBS) and anti-goat (green) IgG-FITC (1:200 in PBS). Sections were then gently washed three times with PBS and cell nuclei counter-stained with DAPI solution (4 mg/ml in deionized water) for 15 min at RT. Both CK18 and B1R/B2R expressions were evaluated and photographed using an IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan) with appropriate excitation/emission filters and in-line Leica camera.
3
Live/Dead Cell Quantification Protocol
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Live/dead assay (ThermoFisher, Burlington ON, Canada) was performed according to manufacturer’s instructions, and as previously reported [26 (link),27 (link)]. Images were captured using an Olympus IX73 inverted fluorescence microscope. Live and dead cells were quantified using Image J software (NIH, v.1.6.0, Bethesda, MD, USA).
4
Immunofluorescence Analysis of NF-κB Pathway
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Paraffin sections were dewaxed and repaired with high pressure antigen, and then 3% hydrogen peroxide was added to remove endogenous enzymes. After blocking with normal fetal bovine serum, NF-κB p65 (ab288751, Abcam), p-IκBα (sc-8404, Santa Cruz Biotechnology), and TOPK (ab280209, Abcam) antibodies were incubated overnight at 4°C. Anti-Human IgG1 H&L (Alexa Fluor® 488) (ab200622, Abcam) or Goat Anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (A-11014, Invitrogen) were used as secondary antibodies. The nuclei were stained with DAPI (Invitrogen). The fluorescence was observed under IX73 inverted fluorescence microscope (Olympus Company, Japan).
5
Real-Time Caspase-3 Activity Assay
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RD cells or HEK 293 cells were seeded on coverslips. To visualize caspase-3 activity in real time, the cells were incubated with 5 μM NucView 488 (Biotium) for 30 min prior to virus infection and kept in the medium throughout the experiment, or added at different intervals as described in the text or figure legends. The cells were then washed three times with ice-cold PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Next, the cells were permeabilized with 0.25% Triton X-100 for 10 min, and blocked with goat serum for 1 h. Finally, the cells were incubated with diluted mouse anti-EV71 antibody followed by goat anti-mouse Alexa 594 secondary antibody (red). The cell images were observed using an Olympus IX 73 inverted fluorescence microscope with cellSens imaging software. The images shown are representative of three independent experiments.
6
Ex vivo Testis Culture for Spermatogenesis
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Ex vivo culture was performed as previously described20 (link),84 (link),85 (link) with minor modifications. Briefly, 1.5% (wt/vol in PBS) agarose gels (Dojindo Laboratory, Kumamoto, Japan) were prepared and submerged in the culture medium αMEM/10% KnockOut Serum Replacement/1% Penicillin–Streptomycin) for >24 h, repeatedly 2–3 times. Whole testes dissected from euthanized neonatal Acr-GFP mice (4 days postpartum) were removed from the tunica albuginea and, without cutting into multiple pieces, gently positioned on the agarose gel in a 12-well culture plate with 500 µL of the above-described medium. The medium was changed once a week for 5 weeks. The culture incubators (APC-30DR; Astec) were supplied with 5% CO2 in the air and maintained at specific temperatures (ranging from 30 to 40 °C), controlled with an accuracy of ±0.2 °C.
The grading of spermatogenesis progression based on Acr-GFP expression was performed visually at the time of weekly medium change, using an IX73 inverted fluorescence microscope (Olympus). Grading was performed unblinded as the observation had to be completed in a short period to minimize the temperature fluctuation.
The grading of spermatogenesis progression based on Acr-GFP expression was performed visually at the time of weekly medium change, using an IX73 inverted fluorescence microscope (Olympus). Grading was performed unblinded as the observation had to be completed in a short period to minimize the temperature fluctuation.
7
Piezoelectric Droplet Generation Protocol
8
Hispolon's Cytotoxic Effects on SCC-9 and HSC3 Cells
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SCC‐9 and HSC3‐ cells were seeded and treated different doses (25, 50, 75, and 100 μM) of hispolon or vehicle control for 24 h. Then, the cells were fixed with 4% paraformaldehyde and stained with 50 mg/mL DAPI staining buffer for 5 min, avoiding light. The fluorescence of cells was analysed by Olympus IX73 inverted fluorescence microscope.
9
Caspase-3 Activation Visualization
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For caspase‐3 activated detection, BioTracker™ NucView® 488 Green Caspase‐3 Dye (MilliporeSigma) was used for analysis. After hispolon treatment, SCC‐9 cells were stained with NucView® 488 substrate stock solution (2 μM) for 30 min, avoiding light. Cells can be observed with green fluorescence by Olympus IX73 inverted fluorescence microscope.
10
Organoid Culture of Alveolar Cells
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Sorted club cells (5 × 103 cells/well) or AT2 cells (2 × 104 cells/well) were mixed with MLg fibroblasts in Matrigel (BD Pharmingen, San Diego, Calif)/basic medium (1:1) that contained DMEM/F12 (Corning, China), 10% FBS, 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich), 100 IU/mL penicillin, 100 µg/mL streptomycin and SB431542 (Sigma-Aldrich). The cell mixtures were then added to Transwell filter inserts (Greiner Bio-One, Kremsmunster, Austria) in 24-well plates containing 410 µL medium. Organoid cultures were maintained in an incubator with 5% CO2 at 37 °C, and the medium was renewed every other day. Organoid cultures were observed through an IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan). Clones with diameters greater than 50 μm were counted, and colony-forming efficiency (CFE) was measured by counting the number of colonies in each well as a proportion of the implanted cells 10 days after seeding. Organoid cultures were embedded in Tissue-Ted optimal cutting temperature (O.C.T.) compound (Sakura, Torrance, Calif) for immunofluorescence or lysed for RNA analyses.
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