Cells were collected from each medium type at days 7, 15, and 20. Cells were washed in PBS and immediately fixed in 10% formaldehyde solution. Each cell sample was then exposed to CD133, CD90, E-cadherin, and beta-catenin primary antibodies. All primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, TX, USA). After a 24-h incubation, the anti-rabbit IgG HRL-F (ab) and 2-PE anti-goat IgG-FITC (Santa Cruz, TX, USA) secondary antibodies were added. Immunostained cells were observed with a fluorescence microscope. For confocal microscopy, cells were fixed in glass chamber slides with 10% paraformaldehyde in PBS for 30 min at room temperature. After two washes with PBS, cells were permeabilized with 100% methanol for 5 min at room temperature. Cells were washed twice with PBS and blocked for 45 min in 5% BSA in PBS. Confocal microscopy studies were carriedout using a laser-scanning microscope from OLYMPUS (Tokyo, Japan).
Laser scanning microscope
Manufactured by Olympus
Sourced in Japan, United States
The Olympus laser scanning microscope is a high-resolution imaging system that uses a focused laser beam to scan specimens and create detailed digital images. The core function of this equipment is to capture and construct microscopic images with exceptional clarity and precision.
Automatically generated - may contain errors
Lab products found in correlation
Dxm1200, by Nikon (3 mentions)
2 pe anti goat igg fitc, by Santa Cruz Biotechnology (2 mentions)
Fluoview software, by Olympus (2 mentions)
Imagej, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cm1850, by Leica (1 mentions)
Hoechst 33258, by Yeasen (1 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Cellstripper, by Corning (1 mentions)
Ldh assay kit, by Nanjing Jiancheng (1 mentions)
Anti brdu antibody, by Abcam (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Alexa fluor labelled secondary antibody, by Cell Signaling Technology (1 mentions)
Goat serum, by Boster Bio (1 mentions)
Ab16911, by Abcam (1 mentions)
Sc374650, by Santa Cruz Biotechnology (1 mentions)
Anti pna, by Vector Laboratories (1 mentions)
Anti cd3 antibody, by Abcam (1 mentions)
Anti rabbit igg hrl f ab 2 pe, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti human e cadherin antibody, by Santa Cruz Biotechnology (1 mentions)
29 protocols using laser scanning microscope
1
Multilineage Stem Cell Characterization
2
Immunohistochemical Analysis of Murine Pancreas
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Mice were sacrificed with CO2 gas and then the abdominal and thoracic cavity were opened. After totally bleeding by cutting off the right atrial appendage, the complete pancreas was stripped down along the duodenum with surgical forceps. Pancreases obtained from mice were fixed in 10% formalin and then embedded in paraffin for sectioning. The paraffinized sections were heated for 15 minutes at 55°C, deparaffinized (2 × 100% xylene for 5 minutes each, 2 × 100% ethanol for 5 minutes each, 2 × 95% ethanol for 5 minutes each, and 70% ethanol for 5 minutes), and then rinsed in ddH2O for 5 minutes. Antigen retrieval was performed by heating the slides at 100°C for 8 minutes in an acidic retrieval solution. The samples were blocked in 3% (w/v) BSA for 15 minutes at RT before incubating at 4°C overnight with primary antibodies against 4‐HNE, Nrf‐2, insulin or HO‐1 diluted in 3% BSA. After being washed, the specimens were incubated in fluorochrome‐conjugated secondary antibody diluted in 3% BSA for 1 hour at RT in the dark. Nuclei were stained with DAPI and then secured with a coverslip. Images were obtained using a laser scanning microscope (Olympus).
3
Lysosomal and Acidic Organelle Staining
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Cells were seeded on coverslips and incubated with 0.5 μM LysoTracker Blue for 2 h or with 5 μg/ml AO for 15 min at 37 °C. After being washed with PBS three times, the cells were sealed with 20% glycerinum (diluted in PBS) and then visualized immediately with an Olympus laser scanning microscope.
4
Immunohistochemical Staining of Tumor Tissues
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Tumor tissues used for IHC staining were measured as described previously [17 (link)]. The primary antibody against HRD1 (1:200) anti-PFKP (1:200), and secondary antibody (1:1000) were used. Diaminobenzidine (DAB) substrate solution was applied before hematoxylin counterstaining. Normal IgG was used for negative control assays. Images were captured and evaluated with a laser scanning microscope (Olympus).
5
Multimodal Lysosomal and Mitochondrial Imaging
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Cells were adjusted to a density of 50000 per cm2, incubated for 48 h, and then washed with cell culture medium twice. For the Superior LysoProbe and LysoTracker co-stain, a solution of Superior LysoProbe I–IV (1 μM) and LysoTracker Green (2 μM) in cell culture medium was added to pre-washed cells and incubated at 37°C for 45 min. For the Superior LysoProbe I–IV and MitoTracker co-stain, a solution of Superior LysoProbe I–IV (1 μM) and MitoTracker (80 nM) in cell culture medium was added to the pre-washed cells and incubated at 37°C for 30 min. To achieve nuclear staining, cells were incubated with 1 μM Hoechst at 37°C for 15 min prior to imaging. Confocal fluorescence imaging studies were performed with an Olympus laser-scanning microscope with a 60× oil-immersion objective lens. Image analysis was performed using Image J (National Institute of Health). For quantitative analysis of fluorescence of the confocal images, the threshold of the images was set to 10, the area of fluorescence was selected with the “create selection” program function, and the fluorescence intensity of the comprehensive image was measured.
6
Chondrocyte Immunofluorescence Assay
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Second-generation chondrocytes were seeded on 6-well plates, by using coverslips, and the cells were exposed to IL-1β (10 ng/ml) or cotreated with 10 ng/ml of IL-1β and limonin at a concentration of 60 μM for in the medium overnight after incubation with a serum-deprived medium for 24 hrs. Next, the coverslips with monolayers of the chondrocytes were washed with PBS for three times, and the cells were fixed with paraformaldehyde (4%) at ~25°C for 15 min and then rinsed with PBS again. Triton X-100 (0.1%) was for the cells and nuclear membrane permeability for 5 min at 25°C. Next, the cells were blocked with BSA (5%) at 37°C for 60 min, washed with PBS, and incubated with primary antibodies: Collagen II (1: 200), MMP13 (1 : 200), p65 (1: 200), and Nrf2 (1 : 200) at 4°C for 24 hrs. And then, the cells were incubated with secondary antibodies (1 : 400) at 25°C for 60 min after being washed with PBS. After that, this was followed by the cells being labeled with DAPI (Invitrogen) for 1 min. The slides were imaged by the Olympus laser scanning microscope, and the fluorescence intensity was evaluated by ImageJ (Bethesda, MD, USA).
7
Confocal and Light Microscopy Imaging
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Confocal images were exported from the Olympus laser-scanning microscope with Fluoview software and stored as tiff files. Conventional light microscopic images were acquired using an Olympus microscope (BX61) attached to a Nikon digital camera DXM1200 and stored as tif files. All compared images were taken using the same intensity and exposure time. All figures were prepared using Photoshop 8.0 graphics software. Only minor adjustments of brightness were made.
8
Immunofluorescence Localization of HOXA5
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Tissues were sectioned in 10 μm thick slices and thawed, mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar, Germany), air-dried, and fixed for 10 min in ice cold acetone. Slides were washed in PBS and incubated for 2 h in a mixture of PBS supplemented with 0.2% Triton X-100 and 0.1% bovine serum albumin, followed by incubation overnight with the primary antibody (1:100). The secondary antibody employed to visualize the localization of HOXA5 is Cy3-conjugated goat anti-rabbit IgG (1:1,000). DAPI was used for staining the nucleus. Visualization was done with a laser scanning microscope (Olympus, Tokyo, Japan).
9
Erythrocytes Phagocytosis by Splenic Macrophages
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To investigate the morphological changes, erythrocytes from mice (n = 3 per group) were collected, labeled with CFSE, plated on confocal dishes (Jet Bio-Filtration Co., Ltd., Guangzhou, China), and captured by FV3000 confocal laser scanning microscope (Olympus, Shinjuku City, Tokyo, Japan). To determine erythrophagocytosis in vitro, splenic macrophages of mice (n = 3 per group) were isolated from enzyme-digested spleen tissues and plated on the confocal dishes. Adherent macrophages were labeled with Hoechst 33258 (1 μg/mL; Yeasen, Shanghai, China) and co-cultured with the CFSE-labeled erythrocytes at a ratio of 1:20 for 2 h. After lysis of the non-ingested erythrocytes, images of macrophages were captured by laser scanning microscope (Olympus, Shinjuku City, Tokyo, Japan). For flow cytometry, splenic macrophages were enriched by plating on the 6-well-plates and phagocytosed the CFSE labeled erythrocytes. Macrophages were harvested by Cellstripper™ (Corning, NY, USA) and a total of 5 × 104 cells were analyzed by flow cytometer (NovoCyte®; Agilent Technologies, Santa Clara, CA, USA) using the FL1 channel (488/530 nm). Unstained cells were used as negative controls, and each group was analyzed in three biological replicates.
10
Cell Death Evaluation by LDH and Fluorescent Staining
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Cell death was evaluated by lactate dehydrogenase (LDH) release and Calcein-AM and PI double staining. For LDH release, LDH released into cell culture supernatants was detected using LDH assay kit (Nanjing Jiancheng Biology Engineering Institute, Jiangsu, China) according to the manufacturer’s instructions. For Calcein-AM (living cell) and PI (dead cell) double staining, 100 μL of 2 μM Calcein-AM and 4.5 μM PI double staining solution were added into each well and incubated at 37°C for 15 minutes. After washing, the cells were imaged under a laser scanning microscope (Olympus Corporation, Tokyo, Japan). The average fluorescence intensity was assessed using Image Pro advanced 6.0 software.
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