Vectashield containing dapi

Fixed and permeabilized HAEs were incubated with anti-CAR (CARex75490, a gift from Joseph Zabner) followed by Alexa Fluor488 anti-rabbit IgG antibody, anti-dsg2 (human DSG2-biotinylated antibody; BAF947, R&D Systems) followed by donkey anti-goat IgG NorthernLights NL557-conjugated antibody (NL001, R&D Systems), or anti-CD46 (C-10) (SC-166159, Santa Cruz Biotechnology) followed by Alexa Fluor488 anti-mouse IgG antibody. Slides were counterstained and mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA).

Frozen sections were blocked in PBS + 0.1% Triton X-100 (PBT) for 1 h. Primary antibodies were diluted in blocking solution (PBT + 1% BSA + 0.15% glycine) as follows: cleaved-Caspase-3 (casp3, D175, lot 43, rabbit, Cell Signaling Technologies, 1:200 dilution), Mbp (SMI 94R-0100 lot E10172EF, mouse, Covance, 1:1000 dilution), GFP (A11122, lot 1891900, rabbit, Invitrogen, 1:1000 dilution), CD45 (14-045-185 C30F11, Rat, Invitrogen, 1:100 dilution) and F4/80, CD11b, CD68 cocktail (MCA497, MCA74G, MCA1957, Rat, Bio-Rad, 1:200 dilution) and incubation performed overnight at 4 °C. After several washes, slides were incubated with secondary antibody (anti-rabbit alexaFluor555, anti-mouse alexaFluor488, anti-rat Cy3 or anti-rabbit alexaFluor488, Invitrogen, 1:500 dilution each) for 2 h before final washing. For dorsal root ganglia (DRGs) cultures, cells were fixed in 4% PFA for 10 min and immunostained using the same procedure with TuJ1 (MMS-435P, lot TU17044, mouse, Eurogentec, 1:1000 dilution) and Mbp (ab40390, Rabbit, Abcam, 1:200 dilution). Secondary antibodies used were as follows: anti-mouse alexaFluor 488 and anti-rabbit alexaFluor 555 (Invitrogen, 1:500 dilution each). Preparations were then mounted using Vectashield containing DAPI (Vector laboratories) and observed using Spinning Disk from Zeiss.

For analysis of cytoophidium, the cells grown in 13-mm round coverslips were fixed with 4% paraformaldehyde and probed with antibodies in an indirect immunofluorescence assay, as previously described (Keppeke et al., 2018 (link); Keppeke et al., 2022 (link)).
Primary antibodies used: rabbit polyclonal anti-IMPDH2 antibody (12948-1-AP, ProteinTech), mouse anti-Myc monoclonal antibody 9E10 (sc-40, Santa Cruz Biotech), and mouse anti-Flag monoclonal antibody clone M2 (F1804, Sigma). Secondary antibodies used: Cy3-conjugated donkey anti-mouse IgG (#715-165-151, Jackson ImmunoResearch) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (#A-21206, Invitrogen). After immunofluorescence labeling, the cells were covered with VECTASHIELD containing DAPI (Vector Labs, USA), and the images were captured either by a fluorescence microscope with 200 × or 400 × magnification (Axio Imager. M2, Carl Zeiss, Germany) or by a confocal microscope (LSM 800, Carl Zeiss, Germany).

Mouse and human cells were grown on glass coverslips, washed in warm PBS, fixed in paraformaldehyde (4% in PBS, 15 min), and then washed in PBS, permeabilized (10 min, 0.5% Triton X-100 in PBS), and blocked (5% BSA in PBS with 0.1% Triton X-100, 30 min). Antibody labeling was performed by addition of 200 µL blocking solution with primary or secondary antibodies and washing with PBS with 0.1% Triton X-100. Samples were mounted in Vectashield containing DAPI (Vector Laboratories, USA). Images were acquired using a Nikon Ti2 microscope, driven by Metamorph (Molecular Devices), equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled with a Prime 95 sCMOS camera (Photometrics) equipped with a 100 x oil-immersion objective lense. Super-resolution images were obtained using the LiveSR module (Gataca Systems). DAPI, Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 647 were sequentially excited. Z-series from the top to the bottom of fibers were sequentially collected for each channel with a step of 0.1–0.3 µm between each frame. Image quantification was performed using National Institutes of Health’s FIJI (Schindelin et al., 2012 (link)).

The cells were seeded on glass coverslips overnight and then fixed in 4% paraformaldehyde for 15 min at room temperature (RT). The fixed cells were permeabilized with 0.1% Triton X-100 and incubated with 5% BSA blocking buffer for 30 min. After washes with PBS, the cells were incubated overnight at 4°C with a rabbit anti-Hsp27 antibody and then for 1 h at RT with CF^TM 488-labeled anti-rabbit secondary antibody (Sigma Chemical Co.). The coverslips were then mounted with Vectashield containing DAPI (Vector Laboratories), and the cells were examined by fluorescence microscopy (Olympus America, Inc.).

Astrocytes isolated from non-Tg and LRRK2-GS mice were treated with α-synuclein for 24 h and then fixed with 4% paraformaldehyde and then permeabilized with 0.25% triton for 10 min. Cells were washed three times in PBS and blocked with 1% BSA for 1 h. Cells were incubated with primary antibodies overnight at 4 °C. Washed cells were incubated with fluorescent secondary antibody (Invitrogen) for 1 h at room temperature. Cover slips were mounted with Vectashield containing DAPI (Vector Laboratories) and viewed on confocal microscope confocal microscope (TCS, DMi8; Leica).