Sp8 navigator confocal microscope

Immunostained organ sections were imaged at high resolution with a Leica SP5, SP8 or SP8 Navigator confocal microscope fitted with a ×10, ×20 or ×40 objective for confocal scanning. Individual fields or tiles of large areas were acquired from cryosections, vibratome or paraffin sections. Large Z-volumes of the vibratome samples were imaged for 3D representation. All images shown are representative of the results obtained for each group and experiment. Animals were dissected and processed under exactly the same conditions. Comparisons of phenotypes or signal intensity were made with pictures obtained using the same laser excitation and confocal scanner detection settings. Fiji/ ImageJ was used to threshold, select and quantify objects in confocal micrographs. Both manual and automatic ImageJ public plug-ins and custom Fiji macros were used for quantification.

DCs were adhered to fibronectin-coated glass-bottom chambers (80827, ibidi GmbH) and fixed with 2% paraformaldehyde in PHEM (PIPES 30 mM, Hepes 20 mM, EDTA 2 mM, MgCl2 1 mM, pH: 6.9) containing 0.12 M sucrose for 15 min at R/T. Cells were then permeabilized with the same solution containing Triton-X100 (0.2%) and treated with Fc-block (anti-CD16/CD32) in blocking buffer containing 3% BSA and human γ-globulin 50 µg/mL in PHEM for 30 min at RT. Mouse monoclonal anti-α-Tubulin conjugated to FITC (clone DM1A, F2168 Sigma Aldrich) and Alexa Fluor 647-conjugated Phalloidin (A22287 ThermoFisher Scientific) were incubated in the same solution for 2 h at RT. Chambers were washed in Tris-buffered saline (pH: 7.4); upon completion of staining, cells were imbibed in Prolong Gold mountant medium with DNA stain DAPI (P36931 ThermoFisher Scientific). A series of fluorescence and brightfield frames were captured using a Leica SP8 Navigator Confocal Microscope equipped with a pulsed white light laser (WLL, range 470-670 nm) and an HC PL Apo CS2 100x/1.4 OIL objective. Images were acquired at room temperature (25 °C) with hybrid detectors and processed with the accompanying Application Suite X software (LAS X, 3.5.2. 18963; Leica Microsystems GmbH) and Image J software (http://rsbweb.nih.gov/ij/).