According to an established method (Nahidiazar et al., 2015 (link)), cells for superresolution microscopy were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, blocked with PBS containing 5% BSA (Sigma-Aldrich), and subsequently incubated with primary and secondary antibodies with washing steps in between. Imaging was performed with a Leica SR-GSD microscope (Leica Microsystems) equipped with 405-nm/30-mW, 488-nm/300-mW, and 642-nm/500-mW lasers, with samples immersed in OxEA buffer (Nahidiazar et al., 2016 (link)). A 160× oil-immersion objective (NA 1.47) and an EMCCD iXon camera (Andor Technology) were used to collect images. Between 10,000 and 50,000 frames were collected, with a frame rate of 100 Hz. All the datasets were analyzed with the ThunderSTORM module of ImageJ software (Ovesný et al., 2014 (link)).
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Super-resolution Imaging of Fixed Cells
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Corresponding Organization : The Netherlands Cancer Institute
Other organizations : Huygens Institute for History and Culture of the Netherlands, Leiden University
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Variable analysis
independent variables
- Fixation of cells in 4% paraformaldehyde
- Permeabilization of cells with 0.2% Triton X-100
- Blocking with PBS containing 5% BSA (Sigma-Aldrich)
- Incubation with primary and secondary antibodies
- Imaging of samples using a Leica SR-GSD microscope
- Collecting between 10,000 and 50,000 frames at a frame rate of 100 Hz
- Analyzing the datasets with the ThunderSTORM module of ImageJ software
- Use of a 160× oil-immersion objective (NA 1.47)
- Use of an EMCCD iXon camera (Andor Technology)
- Immersion of samples in OxEA buffer (Nahidiazar et al., 2016)
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