Cells (0.3‐1 × 105) were seeded on microscopy chamber wells (1 µ‐Slide 8 well, Ibidi) coated overnight with poly‐L‐Lysine (1 mg/ml, 4°C) and washed. The attached cells were fixed with 4% paraformaldehyde in PBS (2 min at 37°C), washed with staining buffer, and mounted with ProLong Diamond antifade mountant (Molecular Probes).
Where needed, intracellular staining was performed after permeabilization in seeded, fixed cells as described above.
Multidimensional microscopy was performed on a Leica AF6000 LX system microscope (Mannheim, Germany); images were captured using a 40.0 × 0.75 DRY objective lens with the Hg‐arc lamp.
Confocal microscopy analysis was performed in a Leica TCS‐SP2‐AOBS‐UV ultraspectral confocal microscope. Immunofluorescence analysis was done using a 63 × 1.4 objective lens at 0.5 µm intervals. Signals from different fluorescent probes were taken in parallel. Image processing was performed using the ImageJ 1.54p NIH public software and Adobe Photoshop (Adobe Systems, Mountain View, CA). Manders thresholded colocalization coefficients (M1, M2) were calculated using the ImageJ 1.54p NIH software.
Where needed, intracellular staining was performed after permeabilization in seeded, fixed cells as described above.
Multidimensional microscopy was performed on a Leica AF6000 LX system microscope (Mannheim, Germany); images were captured using a 40.0 × 0.75 DRY objective lens with the Hg‐arc lamp.
Confocal microscopy analysis was performed in a Leica TCS‐SP2‐AOBS‐UV ultraspectral confocal microscope. Immunofluorescence analysis was done using a 63 × 1.4 objective lens at 0.5 µm intervals. Signals from different fluorescent probes were taken in parallel. Image processing was performed using the ImageJ 1.54p NIH public software and Adobe Photoshop (Adobe Systems, Mountain View, CA). Manders thresholded colocalization coefficients (M1, M2) were calculated using the ImageJ 1.54p NIH software.