40,000 cells were seeded on PLL-coated coverslips and allowed to adhere for 24–36 h. Cells were fixed using 1% (v/v) formaldehyde and incubated for 15 min at room temperature, washed three times with 1X PBS for 5 min each, and permeabilized with 500 μL of 0.3% (v/v) Triton-X100 for 5 min at room temperature. The coverslips were then immersed in 250 μL of 0.05% (v/v) Congo Red (Sigma; Cat# C6277) solution for 15 min, followed by four cycles of 2 min rinsing with 500 μL of ddH2O. The coverslips were then transferred to humidified chambers and nuclear counterstained with 100 μL DAPI, incubated for 4 min, and mounted on microscope slides using DAKO mounting reagent. Images were acquired using a C2+ Confocal microscope with a Plan-Apochromat TIRF ×100 oil objective (NA 1.45) and NIS-Elements AR software (Nikon).
Plan apochromat tirf 100 oil objective
Manufactured by Nikon
The Plan-Apochromat TIRF ×100 oil objective is a high-performance microscope objective designed for Total Internal Reflection Fluorescence (TIRF) microscopy. It features a numerical aperture of 1.46 and a magnification of 100x, making it suitable for applications that require high-resolution imaging of samples near the surface of a coverslip.
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3 protocols using plan apochromat tirf 100 oil objective
1
Congo Red Staining for Amyloid Detection
2
Congo Red Staining for Amyloid Detection
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40,000 cells were seeded on PLL-coated coverslips and allowed to adhere for 24–36 h. Cells were fixed using 1% (v/v) formaldehyde and incubated for 15 min at room temperature, washed three times with 1X PBS for 5 min each, and permeabilized with 500 μL of 0.3% (v/v) Triton-X100 for 5 min at room temperature. The coverslips were then immersed in 250 μL of 0.05% (v/v) Congo Red (Sigma; Cat# C6277) solution for 15 min, followed by four cycles of 2 min rinsing with 500 μL of ddH2O. The coverslips were then transferred to humidified chambers and nuclear counterstained with 100 μL DAPI, incubated for 4 min, and mounted on microscope slides using DAKO mounting reagent. Images were acquired using a C2+ Confocal microscope with a Plan-Apochromat TIRF ×100 oil objective (NA 1.45) and NIS-Elements AR software (Nikon).
3
Live-Cell Microscopy Imaging Protocol
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Log phase cells were collected via centrifugation, washed once with dH2O and resuspended in dropout medium before mounting on a slide for image acquisition63 (link),64 (link). Asynchronous cells were subjected to live cell microscopy. Single plane images were taken with a Nikon C2+ Confocal Microscope using a Plan-Apochromat TIRF ×100 oil objective (numerical aperture 1.45) and processed with NIS-Elements AR (Nikon). Images were captured with excitation wavelengths of 405 nm and 488 nm with a 30–40 nm pinhole.
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