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- Fig. 1.
Proper paraformaldehyde fixation preserves the architecture of the actin cytoskeleton and is compatible with high-quality SMLM. HeLa (A-C) and Cos-7 (D-F) cells fixed with paraformaldehyde (PFA) dissolved in PBS (A and D), PFA in PEM buffer (B and E) or glutaraldehyde (GA) in cytoskeleton buffer (C and F). All cells were stained with Alexa Fluor-647-labelled Phalloidin and imaged in parallel as described in the Materials and Methods. Representative SMLM images (left) and close ups of the boxed regions (right) are shown. Scale bar A-F: 10 µm, scale bar close ups: 1 µm.
- Fig. 2.
Proper PFA fixation preserves small actin fibers as faithfully as GA fixation. (A) PFA-PEM fixation preserves small actin fibers as faithfully as GA fixation. Individual actin fibers (5 to 10 fibers per condition) were manually segmented and their brightness profiles were plotted and fitted to a Gaussian distribution as described in Materials and Methods. Representative fibers, distribution profiles with Gaussian fit and FWHM are shown. Scale bar: 300 nm. (B) Only PFA-PEM and GA fixation preserve very thin actin fibers. Distribution profiles with Gaussian fit and FWHM of the thinnest detected fibers are shown for each fixation method. Scale bar: 300 nm. (C) Localization precision is not affected by the fixation method. Localization precision of images of actin obtained after different fixations was calculated using the Thunderstorm plugin (Ovesny et al., 2014) of Fiji (Schindelin et al., 2012). Bar graph shows localization precision and intensity (mean±s.d.) as obtained from five independent images (PFA-PBS fixation), seven independent images (GA fixation) and nine independent images (PFA-PEM fixation) (one-way ANOVA was employed to compare the obtained results and no significant differences were found).
- Fig. 3.
Paraformaldehyde fixation, but not glutaraldehyde fixation, enables detection of actin-binding proteins within lamellipodia and ruffles. HeLa cells were stimulated with epidermal growth factor (EGF) (100 ng/ml) for 3 min and fixed with either glutaraldehyde (GA) in cytoskeleton buffer (A) or paraformaldehyde (PFA) in PEM buffer (B) and then stained with either anti-mDia1 (mDia1) or anti-WAVE2 (WAVE2) antibodies or mock stained. All samples were incubated with Alexa Fluor-647-labelled Phalloidin and secondary goat anti-mouse antibodies (anti mouse IgG) labelled with Alexa Fluor-532. Membrane ruffles were imaged in EPI mode. Representative SMLM images show the actin cytoskeleton in red and the actin-binding proteins mDia1 and WAVE2 in green. Dashed white areas depict the region of interest used for the quantification of green particles. Scale bar: 1 µm.
- Fig. 4.
Paraformaldehyde and glutaraldehyde fixation allow comparable detection of focal adhesion proteins. HeLa cells were fixed with either glutaraldehyde (GA) in cytoskeleton buffer (A) or paraformaldehyde (PFA) in PEM buffer (B). Cells were stained for Vinculin or Paxillin followed by Alexa Fluor-532-labeled secondary antibodies along with Alexa Fluor-647-labeled Phalloidin. Basal membranes were imaged in TIRF mode as described in the Materials and Methods. Representative SMLM images depict F-actin in red and focal adhesion proteins in green. Dashed white boxes in the Paxillin and the Vinculin images mark the position of the close ups shown on the right. Scale bar: 1 µm.
- Fig. 5.
Paraformaldehyde fixation, but not glutaraldehyde fixation, enables faithful detection of clathrin-coated structures. HeLa cells were serum starved overnight and then stimulated with EGF (100 ng/ml) for 5 min and fixed with PFA in PEM buffer (A) or glutaraldehyde (GA) in cytoskeleton buffer (B). Cells were stained with anti-clathrin heavy chain (CHC) antibodies and goat Alexa Fluor-647-labeled secondary antibodies. Clathrin-coated structures (CCSs) at the basal plasma membrane were imaged in TIRF mode as described in the Materials and Methods. Representative TIRF images (left), SMLM images (middle) and SMLM close ups (right) corresponding to the dashed white areas are shown. Scale bar: 1 µm. (C) GA fixation perturbs both circular and elongated CCSs. Size of circular and elongated CCSs was obtained by manual segmentation as explained in the Materials and Methods. Scatter plots depict each CCS found in four independent images as a color-coded circle. Note that GA causes a dramatic reduction in the number of CCSs and that the few remaining ones have a smaller size compared to that of the PFA-fixed samples. Pearson coefficients and R2 of the correlation between area and circularity were obtained using GraphPad Prism (GA fixation: P <0.0001; PFA-PEM fixation: P <0.0001). (D,E) Area covered by plaques (D) and number of pits (E) are highly reduced in cells fixed with GA. The area and number of particles are expressed as mean±s.e.m. t-test was calculated with GraphPad Prism (D, P=0.0025; E, P=0.0065, n=3).
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