Tcs sp8 sted 3 confocal microscope

Indirect immunofluorescence assays were performed as previously described (19 (link)). The primary antibody used was anti-β-catenin (Cat. No. 16051; Abcam) at 1 μg/ml dilution, and FITC-conjugated Pierce™ goat anti-rabbit IgG (H + L) secondary antibody (Cat. No. 31635; Thermo Scientific) was used at 1:50 dilution. Cells grown on coverslips were mounted on glass slides in SlowFade™ Gold Antifade Mountant w/DAPI (Cat. No. S36939; Invitrogen). The images were captured on Leica TCS SP8 STED 3× confocal microscope using Leica Application Suite X 2.0.2.15022 software (Leica Microsystems).

After 48 h from transfection, HEK293, SHSY5Y and SKNBE2 cells were seeded on polilysine coated glass coverslips (Microtech S.R.L) overnight. Coverslips were fixed in 4% paraformaldehyde (10 min), permeated with 0.2% Triton-100 (15 min) and then blocked in 1% bovine serum albumin (30 min). The anti-FGFR1 primary antibody (ab824) was incubated for 90 min. Coverslips were then incubated in goat anti-Mouse IgG (H+L) secondary antibody Alexa Fluor 546 (Invitrogen A-11030) for 45 min. Coverslips were then stained with DAPI (Sigma) for 10 min. Slides were mounted with Mowiol® 4-88 (Sigma-Aldrich, 81381) and visualized using a Leica TCS SP8 STED 3× confocal microscope (Leica Microsystems CMS GmbH).

Isogenic control and Olfml3^-/- microglia were cultured on sterile glass coverslips treated with fibronectin. Cells were fixed using 4% (w/v) paraformaldehyde (Millipore Sigma, Burlington, MA, USA), washed three times for 5 min at RT, and permeabilized with 0.1% Triton X-100-Tris-buffered saline (TBST) for 15 min at RT and blocked for 2 hours at RT with normal goat serum (5% w/v) and bovine serum albumin (1% w/v) in TBST. Cells were incubated in primary antibody solution (mouse monoclonal anti-TMEM119 (BioLegend, San Diego, CA, USA #853302; 1:1000) in fresh blocking buffer) overnight at 4 °C. Cells were washed three times for 5 min at RT and incubated in secondary antibody solution for 1 hour at RT (IgG (heavy and light) anti-mouse Alexa Fluor 555 (Molecular Probes, Invitrogen, Carlsbad, CA, USA; 1:1000) in fresh blocking buffer). Cells were washed three times for 5 min at RT and mounted with Vectashield with 4′5-diamidino-2phenylindole (DAPI) (Vector Labs, Burlingame, CA, USA). Images were captured via Leica TCS Sp8 STED 3× confocal microscope.

Worms were mounted on 3% agar pads (in M9 (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, and 1 M MgSO₄)) in a 3 μl drop of M9 containing 25 mM sodium azide (NaN₃, Sigma-Aldrich). Images were acquired using a Leica TCS SP8 STED 3× confocal microscope at 63× x, 40×, or 20× resolution and Z stacks and intensity profiles were generated using Fiji (ImageJ) (Schneider et al., 2012 (link)).

To gain in confocal imaging resolution, we undertook STED microscopy imaging followed by deconvolution treatment on spinal cord microtome sections from a combination of Tg(UAS:TagRFP-CAAX;cmlc2:eGFP) and Tg(pkd2l1:Gal4) transgenic lines carrying the espin^icm26 mutation. The original IHC protocol [21 (link)] was adapted using the goat antirabbit IgG Alexa Fluor 594 secondary antibody (#A-11012; ThermoFisher) diluted to 1/200 to reveal the membrane-tagged TagRFP labeling in CSF-cNs in a STED-stable manner. The ZO-1 staining to outline the central canal was kept unmodified. Imaging of both ZO-1 and TagRFP-CAAX was carried out on a Leica TCS SP8 STED 3× confocal microscope using a 93× glycerol objective. ZO-1 labeling was excited with a white laser at 488 nm (3.5%) and detected on a HyD detector from 500 to 550 nm. TagRFP-CAAX labeling was excited with a white light laser at 590 nm (8.5%), depleted with a 775 nm depletion laser (80%), and detected on a HyD detector from 600 to 650 nm, gated from 0.6 to 5.37 ns. Images were then processed by deconvolution using Huygens software (SVI, the Netherlands). To assess the vertical extension of CSF-cN apical extensions in espin wild-type and mutant fish, we drew polygons outlining the apical extension and fitted an ellipse to extract the “minor” measurement using the polygon tool in Fiji.sc/ [68 (link)].