The bladders and kidneys were aseptically harvested, and embedded in OCT compound with liquid nitrogen. Frozen sections (5 μm) were cut and air-dried at room temperature for 20 min and fixed with cold acetone for 10 min. Then the tissues were immediately submerged into methanol for 20 min and 3% hydrogen peroxide in methanol for 10 min. After rehydration in PBS, sections were blocked with 5% BSA for 1 h, incubated with F4/80 antibody (Abcam, ab6640, 1:100), CD36 antibody (Proteintech, 18836-1-AP, 1:100) in blocking buffer overnight at 4°C. After that, coverslips were washed five times with PBS, and incubated with FITC-labeled secondary antibody (Proteintech) or Alexa Fluor 488/594-labeled second antibody (Proteintech) for 1 h. Finally tissue sections were counterstained with DAPI for nuclei visualization. Images were acquired using the OLYMPUS IX73 fluorescent microscope (Shinjuku, Tokyo, Japan).
Ix73 fluorescent microscope
Manufactured by Olympus
Sourced in Japan, United States
The Olympus IX73 is a fluorescent microscope designed for live-cell imaging and advanced microscopy applications. It features high-quality optics, automated functions, and compatibility with a range of imaging techniques. The IX73 enables users to visualize and analyze fluorescent-labeled samples with precision and clarity.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Amaxa 4d nucleofector, by Lonza (4 mentions)
Sf cell line 4d nucleofector solution sf, by Lonza (4 mentions)
Alexa fluor 488 594 labeled second antibody, by Proteintech (1 mentions)
Fitc labeled secondary antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole dapi mounting medium, by Santa Cruz Biotechnology (1 mentions)
Green 5 fluorescein dutp, by Empire Genomics (1 mentions)
Tecnai g2 20 tem, by Thermo Fisher Scientific (1 mentions)
Npflex, by Bruker (1 mentions)
Facsaria 2, by BD (1 mentions)
Lipofectamin 3000, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Plys1 flag mitogfp ha, by Addgene (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ix71 fluorescent microscope, by Olympus (1 mentions)
Deltavision elite system ix71, by Cytiva (1 mentions)
Lamp1, by Merck Group (1 mentions)
Fluorescence activated cell sorting (facs), by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
27 protocols using ix73 fluorescent microscope
1
Immunofluorescence Staining of Kidney and Bladder
2
Bone Marrow Cell Chromosome Y Assay
3
Synthesis and Characterization of EYO Carbon Quantum Dots
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The EYO carbon quantum dots (EYO CDs) were produced in a digital muffle furnace from Beijing Zhongke Aobe Technology Co., Ltd (Beijing, China). Transmission electron microscopy (TEM) Images of EYO CDs were taken using a JEN-1230 electron microscope at an accelerating voltage of 100 kV (Japan Electron Optics Laboratory) and a Tecnai G2 20 TEM (FEI Company, USA) at an accelerating voltage of 200 kV. Fluorescence images were acquired using an OLYMPUS IX73 fluorescent microscope (Tokyo, Japan). Pure water was produced using a Great Wall Scientific Industry circulating water-type multi-purpose vacuum pump (Henan, China). The UV-Vis spectroscopy was performed using a CECIL instruments spectrophotometer (Cambridge, United Kingdom). Fourier transform infrared (FTIR) spectroscopy was performed using a Thermo spectrometer (California, USA).
4
Visualizing Instability Patterns on Gel Surfaces
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To visualize the instability patterns distributed on the gel surfaces, we used an Olympus IX73 fluorescent microscope (Tokyo, Japan). The microscope can also work as an interferometer with a monochromatic filter (532/25 nm VIS Band Pass Filter) (Optolong Optics CO., Ltd., Kunming, China). To analyze the 3D morphology of the instabilities, we further carried out white-light interferometric measurements by using a surface profiler (Bruker NPFLEX, Billerica, MA, USA). The instrument could precisely reconstruct the 3D surface profiles and measure the average roughness and maximum vertical deformations of the substrates.
5
Cytotoxic Extract Screening Using AO/EB Staining
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The cytotoxic extracts were assessed using the AO/EB staining technique as previously described by Ribble et al. [16 (link)]. The cells were seeded at a concentration of 100,000 cells per well in a six-well plate and incubated for 24 hours at 37°C before treatment with the active fraction LF1 at varying concentrations (5, 15 and 25 μg/ml). Following an incubation period of 24 hours, the cells were detached and pelleted. The supernatant was removed and the cells were subsequently stained with the prepared dye mixture (25 μl cold PBS and 2 μl EB/AO dye mixed in a 1:1 ratio). The stained cell suspension was transferred onto a clean glass slide and covered with a coverslip. The morphological changes relative to the untreated control was observed using the narrow blue excitation filter on an Olympus IX73 fluorescent microscope (Olympus Corporation, Shinjuku, Tokyo, JPN). The images were photographed at ×400 magnification.
6
Mitochondrial Protein Tagging in HK-2 Cells
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HK‐2 cells were transfected with pLYS1‐FLAG‐MitoGFP‐HA (#50057; Addgene, Cambridge, Massachusetts), using Lipofectamin3000 (L3000‐001; Invitrogen, Thermo Fisher Scientific) according to the manufacturer's protocol. Transfected cells were selected with puromycin (1 μg/μL; P8833, Sigma Aldrich). Antibiotic‐resistant cells were then sorted for high GFP expression using FACSAria II (Becton Dickinson, Franklin Lakes, New Jersey), were plated, and MitoGFP expression was validated using a IX‐73 fluorescent microscope (OLYMPUS).
7
Quantifying Circulating Tumor Cells
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Fluorescence levels were assessed with excitation/emission at 485 nm/505 nm for GFP, 555 nm/580 nm for Alexa Fluor 555 and PKH26, 650 nm/665 nm for Alexa Fluor 647, and 358 nm/451 nm for DAPI using a IX73 fluorescent microscope (Olympus, Tokyo, Japan), and cells were photographed and counted by viewing the monitor. High‐resolution image acquisition was accomplished using an EPSON personal computer (SEIKO EPSON, Suwa, Japan). GFP (+)/CD45(−) cells were counted as CTC and were captured by automatically manipulated glass pipettes using the MMI CellEctor system (Tomy Digital Biology, Tokyo, Japan). Images were processed for contrast and brightness using Adobe Photoshop CS2 software (Adobe, San Jose, CA, USA) and were observed by individuals blinded to the clinical status of the patients.
8
Intracellular Ca2+ Imaging with Fluo-4 AM
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Intracellular Ca2+ levels were visualised using fluorescent microscopy and Fluo-4 AM permeant dye (Thermo Fisher Scientific). After incubation with Gd3+ (100 μM) and CP-154526 (50 μM), cells were incubated within media containing 1 µM Fluo-4 AM and 5 μg/ml Hoechst 33342 for 30 minutes at 37 °C before being washed twice with HEPES buffered saline solution and imaged using an Olympus IX73 fluorescent microscope.
9
Immunofluorescence Imaging of Irradiated NCI-H460 Cells
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Twenty‐four hours post‐plating in 35 mm glass‐bottom microwell plates, NCI‐H460 cells were irradiated, and after 48 h, fixed with 4% paraformaldehyde and 0.1% glutaraldehyde, washed and permeabilized by 0.2% Triton‐X100 (Merck), followed by overnight incubation with chicken anti‐vimentin polyclonal antibody (Abcam, Tel Aviv, Israel; cat# 24525) in blocking solution (8% BSA, 0.1% Triton‐X100) and then Alexa Fluor 647‐conjugated secondary anti‐chicken IgG antibody (Invitrogen). Cells were then stained with phalloidin‐TRITC and DAPI. Images were acquired with an automated inverted microscope (DeltaVision Elite system IX71 with resolve 3D embedded imaging software; Applied Precision/GE Healthcare, Issaquah, WA, USA) using a 60×/1.42 oil objective (Olympus). Further image processing was performed using imagej (NIH Imaging Software) [15 (link)].
For bright‐field light microscopy imaging, cells were viewed by UPLFLN PH 10×/0.30 and 20×/0.50 objectives on an Olympus IX73 fluorescent microscope equipped with a DP73 camera or an Olympus IX71 fluorescent microscope equipped with a DP70 camera. Images were captured with Olympuscellsens software.
For bright‐field light microscopy imaging, cells were viewed by UPLFLN PH 10×/0.30 and 20×/0.50 objectives on an Olympus IX73 fluorescent microscope equipped with a DP73 camera or an Olympus IX71 fluorescent microscope equipped with a DP70 camera. Images were captured with Olympus
10
Biodistribution of Fluorescent Aptamer in Mice
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The fluorescent-tagged aptamer Alexa 647-HBA7 (1.7 nmol, 30 μg) was delivered to mice via oropharyngeal aspiration. At 4 and 24 h after aptamer administration, mice were anesthetized with ketamine/xylazine injection, blood was drawn for plasma from descending aorta, the lung was infused through tracheal with PBS by 20 cm H2O gravity, and BALF was collected by passive drainage as described previously.50 (link) After perfusing the lung with PBS, the left lobe was dissected, and half was frozen in liquid nitrogen, while the other half was placed in 4% PFA. The right lung was inflated and fixed with 4% PFA. Heart, liver, spleen, kidney, and brain were removed and divided, with half being flash-frozen in liquid nitrogen and half fixed in 4% PFA. After 5 h of fixation, tissues were transferred into 30% sucrose in PBS for 24 h at 4°C. Frozen 10 μm sections were sliced with a cryostat and mounted onto charged slides. The distribution of Alexa 647-HBA7 was observed under an IX73 fluorescent microscope (Olympus, Waltham, MA, USA), and images were acquired. HBA7 retention in the BALF was estimated via fluorescence intensity measurement at 630 nm. HBA7 retention in the lung was determined by the recovery of HBA7 from lung tissue and BALF via RT-PCR as previously described.52 (link)
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