Axio observer 7 microscope

For immunofluorescence staining, the tissue was fixed in 4% PFA at 4°C until tissue embedding. Paraffin-embedded sections were de-paraffinized and rehydrated. Sections were permeabilized with 0.5% Triton-X in PBS and blocked with PBS 0,5% Triton X-100 and 10% serum and stained with rabbit anti-DCLK1 (Abcam) followed by donkey anti-rabbit antibody coupled to Alexa Fluor 555 (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Images were captured on a Zeiss Axio Observer 7 microscope and analysed with Zen software (Zeiss). For tuft cell numbers, five to ten representative villi were counted on four independent images per mouse using ImageJ.

Cells were seeded with density 1,500 cells/ well on the eight well chamber Nunc^® Lab-Tek^® Chamber Slide™ system (Sigma, C7182-1PAK) overnight. Cells were fixed in 4% formaldehyde (Thermo Fisher, 28908) for 10 min at RT, permeablized with 0.1% Triton in PBS for 15 min at RT, and blocked with the manufacturer’s blocking agent (Duolink™ In Situ Red Starter Kit Mouse/Rabbit (Sigma, Cat# DUO92101-1KT) for 1 h followed by incubation with paired primary antibodies: Aurora kinase A (1F8) Mouse mAb (CST 12100; 1:100), Myc Rabbit Ab (CST 13987; 1:100); and GSK-3β Rabbit Ab (27C10) (CST 9315; 1:100). PLA detection was performed in accordance with the accompanied instructions manual. Images were captured by Axio Observer 7 microscope (Zeiss) using 10× objective (0.45 N.A) Image analyses were performed with custom Matlab scripts (ver. R2019b).

U2OS 2-6-3 cells containing 200 copies of a LacO-containing cassette were plated on an 18-mm glass coverslip. The next day the cells were co-transfected with lipofectamine 2000 (Invitrogen) and plasmid DNA for 6 h at 37 °C. Next the medium was replaced with DMEM +/+ and incubated overnight at 37 °C. Prior to the UV-C micro-irradiation, the medium was replaced with CO₂-independent Leibovitz L15 medium (Thermo Fisher Scientific) and cells were incubated with 10 µM of PARG inhibitor (Sigma) for 30 min. If indicated the cells were additionally incubated with 10 µM Olaparib. UV-C laser tracks were made using a diode-pumped solid-state 266-nm Yttrium Aluminum Garnet laser (average power 5 mW, repetition rate up to 10 kHz, and pulse length 1 ns). The UV-C laser is integrated into a UGA-42-Caliburn/2 L Spot Illumination system (Rapp OptoElectronic). Micro-irradiation was combined with live-cell imaging in an environmental chamber set to 37 °C on an all-quartz widefield fluorescence Zeiss Axio Observer 7 microscope, using a ×100 (1.2 NA) ultrafluar glycerol-immersion objective (UV-C). The laser system is coupled to the microscope via a TriggerBox, and a neutral density (ND-1) filter blocks 90% of the laser light. An HXP 120-V metal-halide lamp was used for excitation. Images were acquired in Zeiss ZEN and quantified in ImageJ.

A 3.5 cm culture dish for monitoring cell growth of P0 WBM culture was placed in the chamber of an inverted Axio Observer 7 microscope (Carl Zeiss) at 37 °C and 5% CO₂. Tiled images with z-slices were acquired as 20 per tile every 45 min for 8 days (day 5–13 of P0 WBM culture). The acquired images were stitched and processes with orthogonal projections for maximum intensity using ZEN (Zeiss) software and included as Supplementary Movie 1–3.

To study the microglial activation and migration upon hTau40^WT exposure, N9 microglial cells were exposed to oligomers and aggregates the same as described above. Then, the microglial actin network was studied for active migration by immunostaining with the β-actin antibody (1:250). The microglial Iba1 was analyzed in the Zeiss Axio observer 7 microscope with apotome 2.0. Similarly, the intensity of actin, Iba1, and associated surface area of 3 microglia per field (n = 10 fields) was monitored by ZEN 2.3 software for the identification of migratory growth axis followed by microglial activation. The localization of actin and Iba1 in N9 cells was also observed by immunofluorescence study.

To visualise vesicle and mitochondria transfer along the TNTs, HGF pre-treated cells on coverslips were preloaded with red mitochondrial cytopainter for 30 min following the manufacturer’s instruction and then washed three times with PBS. Separate cell populations were also preloaded with either DiO (5 μM) or red mitochondrial cytopainter for 30 min. Both cell populations were then seeded together at a 1:1 ratio and treated with HGF for 24 h. Coverslips were mounted on a ludin chamber and enclosed in a humidified atmosphere at 37°C with 5% CO₂. Real-time acquisition of images was captured at 30 s–2 min intervals for a duration of 20 min–4 h. Live cells were imaged with Zeiss LSM980-Airyscan confocal microscope using 40x 1.3 NA oil objective or on a Zeiss Axio Observer 7 microscope using a 20x 0.5 NA objective.