The sections stained with DAPI were examined with the observer fluorescence microscope (Zeiss GmbH, Germany). The 10 ×, 20 × and 40 × objectives were used, where with the 40 × immersion oil objective had to be applied to the samples. The microscopy was performed at a wavelength of 358 nm (DAPI, blue channel), 488 nm (green channel), and 594 nm (red channel). The software Zeiss Zen Blue Edition was used for the evaluation and processing of the images (Supplementary Tables 3 , 4 ).
Observer fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Observer fluorescence microscope is a versatile optical instrument designed for observing and analyzing fluorescent samples. It utilizes specialized illumination and detection techniques to capture images of fluorescently labeled specimens, enabling researchers to visualize and study cellular structures, biological processes, and other fluorescent-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab9610, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Fluoromount g mounting medium, by Thermo Fisher Scientific (2 mentions)
Zen blue edition, by Zeiss (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fiji imagej, by Adobe (1 mentions)
Fluo 4 am, by Beyotime (1 mentions)
Axiovision imaging software, by Zeiss (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Filipin 3, by Merck Group (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Acetylated α tubulin, by Merck Group (1 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
A21208, by Thermo Fisher Scientific (1 mentions)
A 11057, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rat, by Thermo Fisher Scientific (1 mentions)
Af1062, by R&D Systems (1 mentions)
Ab92305, by Abcam (1 mentions)
D8417, by Merck Group (1 mentions)
12 protocols using observer fluorescence microscope
1
Fluorescence Microscopy Imaging of DAPI-Stained Samples
2
Immunophenotyping of Embryoid Body-Derived Cells
3
Fluo-4AM Calcium Imaging in NSCs
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NSCs were washed twice and then treated with 1 μM Fluo-4AM (S1060, Beyotime Biotechnology) in culture medium. After a 30-minute incubation at 37°C, the cells were stimulated with rTMS and imaged using a Zeiss Observer Fluorescence Microscope and Axiovision imaging software.
4
Visualization of Autophagic Flux
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In our study, ptf‐LC3 vector (mRFP‐GFP‐LC3 reporter construct) was purchased from Addgene, USA. H838 cells were grown in 35 mm glass‐bottom dishes. On reaching 50% confluence, the cells were transfected with different vectors using Lipofectamine 3000 for 24 hours. Then, the cells in different groups were imaged in phenol red‐free medium using a Zeiss Observer Fluorescence Microscope.
5
Immunofluorescence Staining of MSCs
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MSCs were washed with PBS before being fixed with 1 h treatment of 4% (wt/vol) formalin. Fixed cells were washed twice before being treated with primary antibodies incubated in PBS or permeabilization buffer overnight at 4°C. Following primary antibodies incubations, all cells were washed twice and treated with secondary antibodies for 1 h at room temperature. The secondary antibodies were dissolved in PBS. Cells were then washed twice before being mounted using Fluoroshield with DAPI and glass coverslips. Cells were imaged using a Zeiss Observer Fluorescence Microscope and Axiovision imaging software.
6
Filipin Staining and Ptch1:LacZ Quantification in SCOs
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For filipin staining, SCOs were fixed in 4 % paraformaldehyde (Thermo Fisher Scientific) in 1X PBS for 10 min after one, two, or three days. SCOs were stained in 30 µM filipin III (Sigma/Aldrich) in 1X PBS for 30 min in the dark and mounted in Fluoromount G Mounting Medium (Thermo Fisher Scientific) after washing once with 1X PBS. Images were taken with a Zeiss Observer fluorescence microscope with a 63X objective and oil immersion.
Reporter Gene Assay for Ptch1:LacZ induction Ptch1:LacZ expression levels were quantified 72 h after aggregation of SCOS using the Galacto-Light Plus TM system (Applied Biosciences) according to the manufacturer's instructions. Shortly, SCOs were collected in plastic tubes, washed with 1X PBS, and lysed using lysis buffer (100 mM potassium phosphate pH 7.8, 0.2% Triton X-100). Lysates were incubated with 70 µl Reaction buffer for 30 min in a 96 well plate, followed by an incubation with Accelerator-II for 15 min.
Signal was read in a microplate luminometer for 5 s per well. Ptch1:LacZ levels were normalized to total protein using the Bradford reagent (BioRad).
Reporter Gene Assay for Ptch1:LacZ induction Ptch1:LacZ expression levels were quantified 72 h after aggregation of SCOS using the Galacto-Light Plus TM system (Applied Biosciences) according to the manufacturer's instructions. Shortly, SCOs were collected in plastic tubes, washed with 1X PBS, and lysed using lysis buffer (100 mM potassium phosphate pH 7.8, 0.2% Triton X-100). Lysates were incubated with 70 µl Reaction buffer for 30 min in a 96 well plate, followed by an incubation with Accelerator-II for 15 min.
Signal was read in a microplate luminometer for 5 s per well. Ptch1:LacZ levels were normalized to total protein using the Bradford reagent (BioRad).
7
Cell Lineage Tracking in SCOs
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For cell lineage tracking experiments, cells were incubated with 500 µM CMFDA Green or CMAC Blue (both Invitrogen) in DFNB medium for 45 min at 37 °C and seeded as described above. After 48 h (or respective time points), SCOs were fixed in 4 % paraformaldehyde (Thermo Fisher Scientific) in 1X PBS for 10 min and mounted directly in Fluoromount G Mounting Medium (Thermo Fisher Scientific). Images were taken with a Zeiss Observer fluorescence microscope with a 20X and 10X objective.
8
Chondrocyte Cilia Formation Assay
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Human primary chondrocytes or C28/I2 cells were transfected with IFT88 siRNA or scrambled siRNA (control), trypsinized 24 h later, and then plated on poly-L-lysine-coated coverslips prior to incubation at 37 C for 24 h. The cells were then fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.5% Triton X-100/PBS. After blocking with goat serum at room temperature, the cells were incubated with primary antibody against acetylated a-tubulin (Sigma). The binding of the primary antibody was detected with AlexaFluor 488 conjugated secondary antibody (Cell Signaling) and imaged under a Zeiss Observer fluorescence microscope.
9
BODIPY Staining of H838 and H1703 Cells
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For BODIPY staining, H838 cells and H1703 cells were washed with PBS, and incubated with 1 μM BODIPY 581/591 C11 (Thermo Fisher, USA) for 0.5 h at 37 oC. Then the cells were washed with PBS and analyzed with Zeiss Observer Fluorescence Microscope (Zeiss, Germany).
10
Live cell imaging of autophagy
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The Live cell immunofluorescence microscopy was performed as our previously described [25 (link), 36 (link)]. Briefly, H838 cells and H1703 cells, cultured in 35 mm glass-bottom dishes, were transfected with ptf-mRFP-GFP-LC3 reporter construct using Lipofectamine 3000 (Thermo Fisher, USA) for 24 h. Then, the cells in each group were imaged with a Zeiss Observer Fluorescence Microscope (Zeiss, Germany) in phenol red-free medium.
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