Tcs sp2 aobs apparatus

CLSM observations were performed with a Leica TCS SP2 AOBS apparatus, using a 63x/1.40 NA oil objective and excitation spectral laser lines at 405, 488 and 594 nm. Image acquisition and processing were carried out using the Leica Confocal Software 2.6 rel 1537 (Leica, Wetzlar, Germany) and Adobe Photoshop CS2 1.4.10 software programs (Adobe Systems). Signals from different fluorescent probes were taken in sequential scan settings. Several cells for each labeling condition were analyzed and representative results were reported. Examinations were performed on either unfixed or fixed and permeabilized cells, as previously described [21 (link), 24 (link)]. Flow-cytometry analyses were performed as reported [21 (link), 40 (link)]. Several cells were analyzed for each labeling condition and representative examples are shown.

Three-μm-thick sections in paraffin of human PsA and OA synovia were stained after deparaffination in xilene (5 min, two times), followed by passages in: absolute ethanol (3 min), 95% ethanol in water (3 min), 80% ethanol in water (3 min), 70% ethanol in water, and antigen retrival (5 min at 95°C in 10 mM sodium citrate, pH 6.0). Slides were saturated with blocking buffer (PBS, 0.05% tween 20, 4% BSA) for 1 hour at room temperature. Specimens were stained with a polyclonal rabbit anti-LL37 (Innovagen), rabbit anti-MPO (Abcam), mouse anti-MxA (Novus Bio), monoclonal mouse anti-LL37 (Mab137), polyclonal rabbit anti-human C9 (ATLAS). The following antibodies were used: donkey anti-rabbit IgG AlexaFluor-568 or-647, anti-mouse AlexaFluor-647 and an anti-goat AlexaFluor-488 (Abcam). After washing, slides were mounted in Prolong Gold anti-fade media containing a DNA dye (DAPI) (Molecular Probes). CLSM observations were performed with a Leica TCS SP2 AOBS apparatus, using a 63x/1.40 NA oil objective. Acquisition of images was performed by a Leica confocal software 2.6 (Leica, Germany).

To allow spheroid formation, 1 × 10⁴ cells/ml of melanoma Me 30966 cells were cultured in 96-well plate (Ultra Low Attachment, Costar, Paris, France) in complete cell culture medium until 72 h at 37 °C and 5% CO₂ in continuous rotation. Free AO or Exo-AO derived from macrophages (M ϕ Exo-AO) were then added for 10′, 6 h, 24 h or 48 h. After this time, point cells were washed, fixed with 3% paraformaldehyde (PFA) for 30′ at room temperature, seeded in coverslips and then mounted on the microscope slide by using Vectashield^® mounting medium (Vector Laboratories Inc., Burlingame, CA). CLSM observations were performed on a Leica TCS SP2 AOBS apparatus (Leica Microsystems, Wetzlar, Germany), using excitation spectral laser lines at 488 and 546 nm, and using the confocal software (Leica, Wetzlar, Germany) and Photoshop CS2 (Adobe Systems, San Jose, CA). Signals from different fluorescent probes were taken in sequential scanning mode, several fields were analyzed for each labeling condition, and representative results are shown. Images were obtained by Z-projection of 18–20 optical sections taken from the bottom to the edge of the spheroids.