Directly after antibody staining, the gel where incubated in NHS Ester (Thermo Fisher Scientific catalog number: 46402) and diluted at 10 μg/mL in PBS for 1 hour and 30 minutes at RT on a rocking platform. The gels where then washed 3 times for 15 minutes with PBS-Tween 0.1% then expanded overnight in ultrapure water.
Imaging was performed on a Leica Thunder inverted microscope (Germany) using 63X 1.4NA oil objective with Small Volume Computational Clearing mode to obtain deconvolved images. Three-dimensional stacks were acquired with 0.21 μm z-interval and x,y pixel size of 105 nm. Images were analysed and merged using ImageJ software.
Confocal microscopy was performed on a Leica TCS SP8 (Germany) with a 63×/1.4-NA (numerical aperture) oil immersion objective, using the HyVolution mode20 to generate deconvolved images, with the following parameters: “HyVolution Grade” at max resolution, Huygens Essential as “Approach,” water as “Mounting Medium,” and Best Resolution as “Strategy.”
Imaging was performed on a Leica Thunder inverted microscope (Germany) using 63X 1.4NA oil objective with Small Volume Computational Clearing mode to obtain deconvolved images. Three-dimensional stacks were acquired with 0.21 μm z-interval and x,y pixel size of 105 nm. Images were analysed and merged using ImageJ software.
Confocal microscopy was performed on a Leica TCS SP8 (Germany) with a 63×/1.4-NA (numerical aperture) oil immersion objective, using the HyVolution mode20 to generate deconvolved images, with the following parameters: “HyVolution Grade” at max resolution, Huygens Essential as “Approach,” water as “Mounting Medium,” and Best Resolution as “Strategy.”