ARPE-19 cells were cultured in 6-well plates at a density of 6 × 104 cells per well and transiently transfected with mCherry-eGFP-LC3 plasmid expressing an RFP-GFP-LC3 construct using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. After 72 h of transfection, the cells were treated with Ro 25-6981 for 8 h and then fixed with 4% formalin. Hoechst 33342 staining was performed to determine the degree of A2E accumulation and cellular apoptosis induced by BL irradiation, and the slides were examined using a laser scanning microscope (Nikon, Tokyo, Japan). Additionally, LC3-II levels were measured in samples treated with Baf A1, Ro 25-6981, or both compounds simultaneously for 8 h. Quantification of LC3-II band density was performed using ImageJ software.
Laser scanning microscope
Manufactured by Nikon
Sourced in Japan
The Laser Scanning Microscope is a high-resolution imaging tool that uses a focused laser beam to scan and capture detailed images of samples. It provides a non-invasive way to study the internal structure of various materials and biological specimens with high precision and clarity.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pkh67 fluorescent cell linker kit, by Merck Group (1 mentions)
Coralite594 conjugated goat anti rabbit igg h l secondary antibodies, by Proteintech (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Anti cd68 antibody, by Abcam (1 mentions)
Fluorescein conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Anti brdurd antibody, by Merck Group (1 mentions)
7 protocols using laser scanning microscope
1
Autophagy Modulation in ARPE-19 Cells
2
Visualization of Exosome Internalization in SKOV3 Cells
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The exosomes were marked by PKH67 Fluorescent Cell linker kits (Sigma, St. Louis, MO, US) according to its direction, and the exosomes marked by PKH67 were acquired. A number of (0.5–1) × 105 SKOV3 cells were seeded into 24-well plates and incubated at 37 °C, with 5% CO2. The exosomes marked by PKH67 as well as SKOV3 cells were co-cultured without light for 12 h and washed by PBS for three times, 5 min/time, then fixed by paraformaldehyde for 20–30 min, rinsed by PBS for three times, 5 min/time; the nuclei were stained by 2,4-diamino-5-phenylthiazole (DAPI) (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 5 min, rinsed by PBS for three times (5 min/time), and fixed. The distribution of fluorescence was observed by a laser scanning microscope (Nikon Co., Ltd., Tokyo, Japan).
3
Immunofluorescence Staining of Adherent Cells
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Cells were seeded on coverslips and cultured until 70% confluence, then permeabilized in PBS containing 0.4% Triton, fixed in PBS with 4% paraformaldehyde and blocked with 1% BSA for 1 h. The primary antibody was added and incubated overnight, then incubated with secondary antibody for 1 h and stained with DAPI. The cells were visualized and imaged with a laser scanning microscope (Nikon, Japan).
4
Quantifying Ischemic Boundary Cells
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Images were acquired with the 20× primary objective of a laser scanning microscope (Nikon, Chiyoda, Tokyo, Japan) at a resolution of 1024 × 1024 pixels. The number of double-stained cells in the regions of interest drawn within the striatal ischemic boundary (Figure 1B
5
Immunofluorescent Labeling of Erythrocytes
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Erythrocytes were rinsed three times with PBS, fixed with 4% paraformaldehyde 10 min at RT, permeabilized with 0.01% Triton X-100 for 30 min at RT, and then thoroughly washed with 0.3% BSA in PBS. Fixed samples were blocked with 3% BSA, in PBS at 37 °C for 1 h and then incubated with anti-Band 3 antibody overnight at 4 °C. Samples were then washed several times in PBS and incubated for 1 h at 37 °C with CoraLite594—conjugated Goat Anti-Rabbit IgG (H + L) secondary antibodies (Proteintech, Wuhan, China). Stained erythrocytes were imaged on a laser scanning microscope (Nikon, Tokyo, Japan).
6
Histological Analysis of Atherosclerotic Lesions
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For the necrotic core analysis, aortic sinus sections were stained with hematoxylin and eosin (H&E) (Solarbio, Beijing, China). Then, the slices were observed using an optical microscope. For detecting macrophage-derived foam cells in atherosclerotic lesions, cryostat sections were immunostained with anti-CD68 antibodies (1:250, Abcam) at 4°C overnight and then incubated with the corresponding fluorescein-conjugated secondary antibodies (1:200, Life Technologies, NY, USA) for 1 h. After immunostaining with CD68 antibody, the slides were stained with Bodipy 493/503 (Invitrogen, Karlsruhe, Germany) working solution for 1 h at room temperature. Nuclei were stained with DAPI (Beyotime, Shanghai, China) for 10 min. Confocal imaging was recorded in a laser-scanning microscope (Nikon Instruments, NY, USA). The captured images above were analyzed with ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring, USA).
7
Cell Proliferation Analysis via BrdUrd Staining
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Cells were plated on coverslips (Thermo Fisher Scientific, Waltham, MA, USA ) and allowed to settle for 24 hours, incubated with bromodeoxyuridine (BrdUrd) for 1 h and stained with anti-BrdUrd antibody (Sigma) according to the manufacturer’s instruction. Gray level images were acquired under a laser scanning microscope (Nikon Co., Tokyo, Japan).
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