Eclipse ti2

Immunofluorescence was performed as described previously (Schurmann et al., 2019 ), using 2% (v/v) foetal bovine serum (Life Technologies) in place of normal goat serum. Cells were incubated with primary antibodies against BIN1 (ab54764; Abcam), post-synaptic density 95 (PSD95, D74D3; Cell Signaling), synaptophysin (sc7568; Santa Cruz, TX, USA), glial fibrillary acidic protein (Agilent, ZO334) and microtubule-associated protein 2 (GTX82661; GeneTex) and the appropriate species of AlexaFluor-conjugated secondary antibodies (Life Technologies). Coverslips were mounted onto glass slides using the ProLong Diamond mounting media (Life Technologies). The labelled proteins were imaged under an Eclipse Ti2 inverted Nikon 3D structured illumination microscope, and images were reconstructed using Nikon Imaging Systems Elements software, or a Nikon Eclipse Ti2 inverted microscope with Vt-iSIM scan head and deconvolved using Nikon Imaging Systems Elements software.

For stress pilot experiment (Fig. 3a), mice were anesthetized (Avertin, 2,2,2-Tribromoethanol; T48402, Sigma-Aldrich), transcardially perfused with Ringer (Braun-Melsungen, Germany) for 2 min and 4% formaldehyde for 10 min. Brains were 24 h post-fixed in 4% formaldehyde, cryoprotected in 30% sucrose, frozen and embedded in Tissue-Tek (4583, Sakura-Finetek-Europe, Netherlands). Coronal 30 µm-thick sections (cryostat; Leica-CM1950; Leica-Microsystems, Buffalo Grove, IL, USA) were kept in 25% ethylene glycol and 25% glycerol/PBS. Free-floating sections were blocked (1 h/RT) in 5% normal horse serum (NHS) in PBST (1 × PBS + 0.3% Triton X-100) and incubated with rabbit anti-cFos (226003; Synaptic Systems, Göttingen, Germany) 1:1000 in PBST + 5% NHS overnight/4 °C. After washing with PBS, secondary antibody donkey anti-rabbit IgG-Alexa Fluor 647 (A-31573, ThermoFisher) 1:500 in PBST + 3% NHS was incubated for 2 h/RT. Nuclei were visualized with DAPI (Sigma-Aldrich) 1:5000 for 10 min. Sections were mounted using Aqua-Poly/Mount (Polysciences). Tile scans of hippocampus/hypothalamus were acquired using the 20× air-objective from Nikon-Ti2 Eclipse (Nikon, Tokyo, Japan) and cFos+ cells counted using cell-counter-plugin of FIJI-ImageJ-software. Representative images (1024 × 1024; 1 µm intervals) were taken with Leica-TCS-SP5, then processed with FIJI-ImageJ.

Cells were imaged using TIRF-M based on a Nikon Ti2 Eclipse with a 100×/1.45 objective (Nikon). Excitation was done by DPSS laser at 488 nm. The emission light was captured on an EMCCD camera (Hamamatsu ORCA-Flash 4.0). Scaling was maintained at 130 nm per pixel. Most cells were imaged at 100 ms exposure time.

Fluorescent images of the patterns were acquired with an inverted microscope (Nikon Ti2 Eclipse, Nikon) with an objective 20×/0.75 (Nikon CFI Plan Apo 20X/0.75 Ph DM). Confocal images were taken using inverted confocal microscope Axio Observer 7 (Spectral Detection Zeiss LSM 800) using 40×/1.3 Oil DIC M27 or 63×/1.4 Oil DIC M27 objectives with ZEN 2.3 imaging software. For integrin imaging, Zeiss Axiovert 200M microscope was used with 20× and 40× objective. For pMLC2 imaging, Zeiss LSM 780 microscope was used with 20×/0.8 M27 Plan-Apochromat and 40×/1.20 W Korr M27 C-Apochromat objectives using ZEN 2.1 SP3 software.

For epifluorescence imaging, all Z-stack images were obtained with 0.5 microns between slices. Images were acquired in an epifluorescence microscope (Nikon Ti2Eclipse) with an X60/1.25NA and X100/1.3NA oil immersion objectives for bead and spreading assays, respectively. For confocal microscopy, images were acquired in a Nikon Ti2Eclipse inverted microscope with 60X/1.45NA oil immersion for bead and spreading assays, with a Z-stack of 0.5 microns. For Total internal reflection fluorescence microscopy (TIRFM), images were acquired in Nikon Ti2Eclipse inverted microscope with a 100x/1.50 NA oil immersion lens and an iXON Ultra EMCCD camera at 37°C. B-cells expressing LifeAct-mCherry were plated on Ag-coated glass chambers (Nunc^TM Lab-Tek^TM II). Images were acquired for 30 min at 15 s per frame for spreading assay and for 1 min at 0.75 s per frame for lysosome, proteasome, and actin retrograde flow tracking. For Ayriscan acquisition, images were obtained in the Zeiss LSM880 Airyscan Confocal microscope with a 63X/1.4NA oil immersion lens, with a Z-stack of 0.2 μm. The images were processed using Zeiss Black Zen software and analyzed with FIJI.

Microscopy slides were imaged using an inverted fluorescence microscope (Nikon Ti-2 Eclipse with Crest X-light V2 spinning disk module (disk unit 60 μm), Nikon Europe BV, Amsterdam, Netherlands) with a CFI Plan Fluor 40× oil immersion objective (CFI Plan Fluor 40×/1.30 W.D. 0.24, Nikon Europe BV). Brightfield and fluorescence images were recorded by an Andor Zyla 4.2 Plus USB3 camera in Widefield and Spinning Disk Confocal mode using LED light excitation. HPTS was imaged with excitation at 395 nm and 470 nm and emission at 515 nm using appropriate exciter, emitter, dichroic filter cubes. Liss Rhod PE was imaged with excitation at 550 nm and emission at 595 nm and DY-647P1 with excitation at 640 nm and emission at 698 nm using appropriate exciter, emitter, dichroic filter cubes. Z-Stacks were recorded in confocal mode from top to bottom (below the slide surface) with a step size of 1 μm and 72 steps. Time series were recorded as indicated.