Stellaris 8 falcon τ sted

The labelled gliosomes and slices were analyzed by confocal microscopy using a Leica STELLARIS 8 Falcon τSTED (Leica Microsystems, Mannheim, Germany) inverted confocal/STED microscope. Excitation was provided by a white light laser, selecting the combination of chosen fluorochromes to avoid crosstalk. Detection was performed by three Power HyD detectors. The fluorescence image (1024 × 1024 × 16 bit) acquisition was performed using an HC PL APO CS oil immersion objective 100× (1.40 NA). The pinhole was set to 1 Airy size. Line scanning speed range was 400 Hz. Leica “LAS X application Suite” software package 4.4.0.24861 was used for acquisition, storage, visualization, and 3D analysis.

Imaging on fixed and permeabilized synaptosomes was carried out as previously described [88 (link),92 (link)]. The following antibodies were used: rabbit anti-synaptophysin (1:500; Sigma-Aldrich, St. Louis, MO, USA), mouse anti-GFAP (1:1000; Sigma-Aldrich), mouse anti-oligodendrocyte (RIP; 1:10,000; Millipore Corporation) and mouse anti-integrin-αM (1:25; Millipore Corporation). Anti-rabbit and anti-mouse secondary antibodies (conjugated with Alexa Fluor 488 or 633) were used (1:1000; Life Technologies Corporation, Carlsbad, CA, USA). Images were collected by means of confocal microscopy using an inverted Leica STELLARIS 8 Falcon τ-STED (Leica Microsystems, Mannheim, Germany) inverted confocal/STED microscope. A white light laser was used and optimally tuned to provide excitation of the chosen fluorochromes. Hybrid HyD detectors were used for the detection. An HC PL APO CS oil immersion objective 100× (1.40 NA) was used to collect the images, while the pinhole was set to 1 Airy size. The line scanning speed range was 400 Hz. The Leica “LAS X application Suite” software package 4.4.0.24861 was used for acquisition, storage and visualization. The purity of synaptosomal fraction was assessed by analyzing 5–7 fields from at least three different preparations.