Tcs sp8 x microscope

C2 cells were plated in 100 mm Petri dishes (2 × 10⁶ cells/well), incubated for 18 h before treatment with 50 or 100 μM 4-IPP (24 h) or with 5 mM metformin (120 h). The cells were stained with MitoTracker Deep Red FM (Molecular Probes, Invitrogen, Carlsbad, CA, USA) at a final concentration of 60 nM, then fixed with 4% paraformaldehyde at 37 °C for 15 min, and washed with phosphate-buffered saline. Immunofluorescence staining was performed on 2-μm paraffin-embedded sections, deparaffinised in xylene and rehydrated in graded alcohols. The slides were washed in 0.05 M phosphate-buffered saline, incubated with DAPI 1:35,000 (Molecular Probes) for 10 min, washed again in 0.05 M phosphate-buffered saline and then fixed using Prolong Antifade (Molecular Probes). Confocal laser scanning microscopy was performed using the Leica TCS SP8 X microscope (Leica Microsystems GmbH, Mannheim, Germany).

Confocal laser scanning microscopy was performed using the Leica TCS SP8 X microscope (Leica Microsystems GmbH).

S. epidermidis ATCC 38984 was grown overnight at 37 °C in TSB using a shaking incubator. Subsequently, the bacteria were diluted in TSB supplemented with 5% glucose and 4% NaCl to a final OD600 of 0.1 and incubated in a 12-well microtiter plate containing 18 mm chemically resistant borosilicate glass coverslips for microscopy (Paul Marienfeld GmbH, Germany). After 48 h of incubation, the coverslips were incubated for 15 min with vancomycin at 0.5, 1, 2, 4, or 8 mg/L final concentration or without vancomycin. Subsequently, the coverslips were washed once with PBS, incubated with 0.07 nmol/mL of vancomycin-BODIPY™ FL (vanco-BODIPY) for 15 min (Thermo Fisher Scientific, USA), washed once with PBS to remove any unbound tracer, and fixed with 4% paraformaldehyde. Finally, the coverslips were mounted on microscopy slides. Image acquisition was performed with a Leica TCS SP8X microscope.

Immunofluorescence staining was used to reveal the cellular composition of Matrigel, collagen I and collagen:basement membrane cultures. Samples were fixed in 4% formaldehyde for 1 h, permeabilised with 0.2% Triton-X in PBS for 1 h and blocked with Antibody Diluent (ImmunoLogic, VWRKUD09-999) for 30 min. The following antibodies were used: anti-lysozyme EC 3.2.1.17 (1:200, DAKO, A0099), anti-aldolase B EPR3138Y (1:200, Abcam, ab75751) and anti-Ly6a-APC (1:200, eBioscience, 17-5981-81).
Snap-frozen mouse intestinal tissue (E19, P0 and adult) were cut at a thickness of 10 μm using a cryostat and transferred onto Superfrost microscope slides. Sections were fixed with 4% paraformaldehyde for 10 mins at room temperature, permeabilised with 0.2% Triton-X 100 for 10 mins and blocked with Antibody Diluent for 10 min. Slides were incubated with anti-laminin polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-22901) overnight at 4°C. Images were taken using the Leica TCS SP8 X microscope.

Interaction of ABCA1 and APE1/Ref-1 was visualized using a Duolink II fluorescence kit (Sigma-Aldrich, St Louis, MO, USA) as described previously [15 (link)], with some modifications. HEK293T cells expressing wild type APE1/Ref-1-FLAG [45 (link)] or mutant APE1/Ref-1(K6/7R)-FLAG [46 (link)] grown on glass coverslips were treated with TSA for 1h. After fixation and blocking the cells were incubated with a mixture of goat anti-ABCA1 antibody (1:200) and rabbit anti-APE1/Ref-1 antibody (1:500) overnight at 4 °C. For in situ analysis, conjugated oligonucleotides [PLA probe anti-goat minus (minus strand) plus anti-rabbit antibody (plus strand)] were added and incubated for 1 h at 37 °C. A negative control was prepared by adding anti-mouse IgG. Fluorescence confocal microscopic images of the cells were acquired using excitation/emission wavelengths of 590/670 nm for the PLA signal and 410/470 nm for the DAPI (Sigma-Aldrich, St Louis, MO, USA). Images were acquired with a confocal TCS SP8 X microscope (Leica, Wetzlar, Germany) with 40× oil immersion objective. Z-stack image were collected at 0.4 µm intervals. The obtained images were processed using Leica LAS X software (Leica, Wetzlar, Germany).

Worms were anaesthetised using 2 μM levamisole and mounted on 5% agar pads. Images were collected using an Olympus BX53 or a Leica TCS SP8 X microscope. The GFP intensity of DAF-16::GFP::3xFLAG was measured by ImageJ (1.53t). Background signals were subtracted as reported^{77 (link)}.