Axio observer lsm710 confocal microscope

The cells were fixed with 4 % PFA, and unspecific binding was prevented by incubating cells in a 3 % BSA and 0.5 % Triton X-100 solution (all from Sigma-Aldrich) for 30 min at RT. The cells were incubated with primary antibodies prepared in 0.3 % BSA and 0.1 % Triton X-100, kept overnight at 4 °C, washed with PBS, and finally incubated for 1 h at RT with the corresponding secondary antibody. Antibodies used were as follows: rat monoclonal anti-CD11b (1:600; AbD Serotec), mouse monoclonal anti-acetylated α-tubulin (1:100; Sigma-Aldrich), goat polyclonal anti-Nox1 (1:250; Santa Cruz Biotechnology, Heidelberg, Germany), Alexa Fluor 594 goat anti-rat, Alexa Fluor 488 goat anti-rat, Alexa Fluor 488 donkey anti-goat, and Alexa Fluor 594 rabbit anti-mouse (all 1:200 in PBS, from Life Technologies Ltd). Membrane ruffling was observed using phalloidin, a marker for filamentous actin. The cells were incubated for 2 h with Alexa Fluor 594 conjugated to phalloidin (1:100; Life Technologies Ltd) in PBS, at RT. For nuclear labeling, cell preparations were stained with Hoechst 33342 (2 μg/ml, Life Technologies Ltd) in PBS, for 5 min at RT and mounted in Dako fluorescent medium. Fluorescent images were acquired using an Axio Observer LSM710 confocal microscope (Zeiss) under a 63× oil objective.

The immunoassays were performed as described by us [24 (link)]. Briefly, brain slices were incubated in a blocking solution containing 2% of horse serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Thermo Fisher Scientific) diluted in 0.1 M PBS for 2 h at room temperature. Then, slices were incubated in the following primary antibodies diluted in the blocking solution for 72 h at 4 °C: rat anti-cluster of differentiation molecule 11b (CD11b) (1:500; AbD Serotec, Oxfordshire, UK), mouse anti-GFAP (1:500; BD Biosciences) or rabbit anti-Ago2 (1:100; Cell Signaling). After that, sections were rinsed in PBS and incubated with the respective secondary antibodies and Hoechst- 33342 (1:1000; Life Technologies) diluted in a solution containing 0.3% Triton X-100 in 0.1 M PBS for 2 h at room temperature: Alexa Fluor-488 donkey anti-rat or anti-mouse, and Alexa Fluor-594 donkey anti-rabbit (all 1:500; all Life Technologies). Finally, tissue sections were rinsed in PBS and mounted in Fluoroshield Mounting Medium (Abcam Plc., Cambridge, UK). Representative images were acquired using an AxioObserver LSM 710 confocal microscope (Carl Zeiss) under a 40× oil immersion objective and are provided in Additional file 4.

Imaging was performed on a Zeiss AxioObserver LSM 710 confocal microscope. FACS-purified 14 day zoledronate-expanded γδT cells were incubated with IgG-opsonized, IPTG-inducible GFP-expressing E. coli (Thermo Fisher) for 60 min, placed on ice and fixed. Cells were then fluorescently labeled, deposited on cleaned coverslips and mounted on glass slides using ProLong Gold antifade mountant (Thermo Fisher) and cured in the dark at room temperature for 24 h. Images of cell conjugates were acquired with a 63× Plan-Apochromat oil objective, numerical aperture 1.4. Acquisition was optimized for subsequent deconvolution with Huygens software, using appropriate voxel sizes according to the Huygens Nyquist calculator.