Lsm 800 confocal system

Sample fixation was performed with a solution of 4% of paraformaldehyde in 0.1 M of PBS (Lonza) [35 (link), 36 (link)]. The following steps were performed: cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min; samples blocking with 5% skimmed milk in PBS for 30 min [37 (link)]; primary antibodies (anti-NFκB, 1 : 200, Santa Cruz Biotechnology; anti-MyD800, Thermo Fisher Scientific; anti-DNMT1, 1 : 200, EpiGentek; and anti-p300, 1 : 200, OriGene) incubation for 2 h at room temperature; and finally, secondary antibody (Alexa Fluor 568 red fluorescence-conjugated goat anti-rabbit antibody, 1 : 200, Molecular Probes, Invitrogen, Eugene, OR, USA) incubation for 1 h at 37°C. Cells were stained for 1 h with Alexa Fluor 488 phalloidin green fluorescent conjugate (1 : 400, Molecular Probes) and for 1 h with TOPRO (1 : 200, Molecular Probes) to mark the cytoskeleton actin and nuclei, respectively [38 (link), 39 (link)]. The Zeiss LSM800 confocal system (Zeiss, Jena, Germany) has been used to acquire microphotographs.

Human DPSCs were seeded in 35 mm-ø dish (µ-Dish, ibidi GmbH, Gräfelfing, Germany) and treated for 24 h in culture medium alone (Control, CTRL) or containing 2 mM HEMA, 50 µgmL⁻¹ AS or 2 mM HEMA plus 50 µg mL⁻¹ AS HEMA + AS. After the incubation time, the cells were washed two times with normal external solution (NES, containing in mM: 125 NaCl, 5 KCl, 1 MgSO₄, 1 KH₂PO₄, 5.5 glucose, 1 CaCl₂, 20 HEPES, pH 7.4) and incubated for 30 min at 37 °C with 10 μM of 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher Scientific, Monza, Italy) in NES containing the respective HEMA and AS treatments as above described. Then, the cells were washed twice with NES and observed in NES alone (CTRL) or containing HEMA or AS alone, or HEMA + AS. For each experimental condition, confocal pictures were casually taken utilizing a Zeiss LSM800 confocal system (Carl Zeiss, Jena, Germany), equipped with an inverted microscope Axio-obserber. D1 and an objective W-Plan-Apo 40X/1.3 DIC. Excitation was fixed at 488 nm and emission wavelength setting the filter set over 505–530 nm. The acquisition settings (laser power, photomultiplier gain, pin-hole and offset) were maintained constant among all specimens acquisitions. Off-line image analyses were executed using Fiji distribution of ImageJ software (v. 1.5i.) [61 (link)].

Confocal images of cell-associated fluorescence were acquired using the Zeiss LSM 800 confocal system (Carl Zeiss Microscopy, Germany) via the Airyscan super resolution mode. Three laser lines, 405 nm (blue, for nuclei), 488 nm (green, for leukocyte surface antigens [CD45 or CD4 in liver, LN, and spleen; CD68 in brain] and 647 nm (red, for HIV-1 protein, p24) were used. Blue, green, and red signals were separated by a quad DAPI/FITC/TRITC/Cy5 dichroic beam splitter and further acquired using a Gasp detector. A Plan-Apochromat 63x/1.4 Oil DIC objective was used to visualize multi-color labelled cell/tissue samples. ZEN Blue 2.3 software (Carl Zeiss Microscopy, Germany) was used to generate original images. All images were acquired under the same instrument settings. Signal to noise ratio was accounted for by averaging data all images acquired using the same gain offset, detector, and laser excitation power. Saturated signal was avoided by using the software-controlled range for a minimum pixel saturation.