Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature; permeabilized in 100 mM tris-HCl (pH 7.4), 50 mM EDTA (pH 8.0), and 0.5% Triton X-100; and incubated with the corresponding primary antibodies overnight (see table S2). This was followed by incubation with corresponding fluorescently conjugated Alexa Fluor secondary antibodies for 2 hours at room temperature. Both primary and secondary antibodies were diluted in PBS containing 1% BSA (Sigma-Aldrich) and 0.1% Tween 20 (Sigma-Aldrich). Images were acquired using either a Nikon spinning disk confocal microscope (CSU-W1) or a Zeiss LSM880 Airyscan microscope. See table S2 for antibody information.
Spinning disk confocal microscope csu w1
Manufactured by Zeiss
The Spinning disk confocal microscope (CSU-W1) is a type of confocal microscope that uses a spinning disk to rapidly scan a sample. It allows for fast and efficient imaging of live cells with minimal photodamage. The CSU-W1 provides optical sectioning, increased resolution, and reduced background fluorescence compared to conventional widefield microscopy.
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2 protocols using spinning disk confocal microscope csu w1
1
Immunofluorescence Staining Protocol
2
Immunofluorescence Staining of Cells
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Cells were fixed in 4 % Paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature, permeabilized in 100 mM Tris-HCl pH 7.4, 50 mM EDTA pH 8.0, 0.5% Triton X-100 and incubated with the corresponding primary antibodies overnight (see STAR methods). This was followed by incubation with corresponding fluorescently conjugated Alexa Fluor secondary antibodies for 2 h at room temperature. Both primary and secondary antibodies were diluted in PBS containing 1% BSA (Sigma) and 0.1% Tween20 (Sigma). Images were acquired using either a Nikon spinning disk confocal microscope (CSU-W1) or a Zeiss LSM880 Airyscan microscope. See Table S2 for antibody information.
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