MCF-7 cells were seeded in a complete cell medium to 24-well plates, which contained cover glasses (thickness 1, Assistant, Karl Hecht GmbH & Co KG, Sondheim/Rhön, Germany). After one day, the treatment was performed in a serum-free medium for different time points (5, 15, 30 and 60 s as well as 5, 10, 30 and 60 min). Afterwards, cells were washed twice with phosphate buffered saline (PBS) and fixed by 4% paraformaldehyde for 20 min at 37 °C. To stain the nuclei, the samples were washed three times with PBS and incubated for 15 min with 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI, 0.2 µg/mL, dissolved in PBS, Sigma-Aldrich Kft). After washing, cover glasses were mounted to microscopy slides (VWR International, Debrecen, Hungary) by Mowiol® 4–88 mounting medium (Sigma-Aldrich Kft). Confocal microscopy studies were performed on a Zeiss LSM 710 system (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 40X oil objective and ZEN Lite (Carl Zeiss Microscopy GmbH) software was used for image processing.
Microscopy slides
Manufactured by Avantor
Sourced in Hungary
Microscopy slides are a fundamental tool used in various scientific and medical applications. They provide a stable, transparent surface for mounting and analyzing small samples under a microscope. These slides are typically made of glass or plastic and come in standard sizes to accommodate different microscope stages and applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm710 system, by Zeiss (2 mentions)
Zen lite, by Zeiss (2 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Zen black software, by Zeiss (1 mentions)
Glass coverslip, by Avantor (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Mowiol 4 88 mounting medium, by Merck Group (1 mentions)
5 protocols using microscopy slides
1
Visualizing Nuclear Dynamics in MCF-7 Cells
2
Tissue Preparation for Confocal Imaging
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Fixed tissue was prepared by transcardially perfusing terminally anesthetized mice with phosphate-buffered saline (PBS; Sigma-Aldrich) immediately followed by 4% paraformaldehyde (PFA; diluted in PBS from a 16% stock solution; Electron Microscopy Sciences). The brain was then removed and transferred into PFA solution overnight before being thoroughly rinsed and stored in PBS at 4°C. Fixed brains were then sectioned into 50-μm thick coronal slices on a Leica VT1200S vibratome and collected in PBS. The sections were positioned on microscopy slides (VWR), allowed to dry and mounted with ProLong Diamond (Thermo Fisher Scientific) and #1.5 glass coverslips (VWR). Mounted sections were stored at 4°C until imaged with an Olympus FV10i-DUC confocal laser scanning microscope, using 10×/0.4 (air) or 60×/1.35 (oil) objective. FIJI (NIH, RRID:SCR_002285) was used to adjust images for brightness, contrast, and pseudo-coloring (dx.doi.org/10.17504/protocols.io.kxygx3nrkg8j/v1) .
3
Cellular Localization of Conjugate Compounds
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Cells were seeded to 24-well plates (5 × 104 cells/well) that contained glass coverslips (diameter: 13 mm, VWR Hungary Kft.) one day before the experiment. Cells were treated with the conjugates (12.5 μM dissolved in serum-free medium) for 15 min, 45 min and 90 min. After washing with serum-free medium and PBS, cells were fixed with 4% paraformaldehyde (37 °C, 15 min, Sigma-Aldrich). Nuclei were stained with Hoechst 33342 (0.3 μg/mL, dissolved in PBS, 37 °C, 10 min, Thermo Fisher Scientific). After washing three times with PBS, coverslips were mounted to microscopy slides (VWR Hungary Kft.) using Mowiol 4-88 (Sigma-Aldrich). Images were captured and photographed using a ZeissLSM 710 confocal laser scanning microscope (Carl Zeiss AG, Jena, Germany), and processed by ZEN black software (Zeiss).
4
Fabrication of Silica-Titania Bragg Stacks
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The fabrication of the silica and titania Bragg stack was previously described (39 ). All starting compounds were obtained from Millipore Sigma. Titania nanoparticles were prepared through sol-gel hydrolysis by slowly adding Titanium (IV) ethoxide to 0.1 N HNO3 and stirring at 80 °C for 8 h. After sonication (Branson Ultrasonics, St. Louis, MI), the particles were filtered and diluted with deionized water. Poly(ethylene oxide) (PEG 8,000-10,000) was added to aid the spin-coating process. SiO2 colloids (LUDOX SM30, 30 wt.% aq.) were diluted with distilled water to a ratio of 2:5 using hydrophilic syringe filters (SPARTAN 13, 0.2 μm). Bragg stacks were assembled on microscopy slides (VWR Inc.) treated with oxygen plasma (Diener Femto PCCE, Ebhausen, Germany) prior to assembly. The slides were covered with 200 to 250 µL of the silica suspension and spun-coated for 60 s at 2,500 to 5,500 rpm with an acceleration of 1,500 rpm s−1 using spin coater (WS-650-23, Laurell, North Wales, PA). Samples were then calcined for 30 min at 350 °C (Lindberg/Blue M BF51866A-1; Thermo Fisher Scientific, Waltham, MA). The procedure was repeated for alternating layers of titania and silica for a total of 5.5 bilayers.
5
Immunofluorescence Imaging of Lysosomal Co-localization
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Cells were seeded to cover glass containing (thickness 1, Assistant, Karl Hecht GmbH & Co KG, Sondheim/Rhön Germany) 24-well plates one day prior to the experiment in complete cell medium. Treatment was performed in serum-free medium for the indicated incubation time. Cells were washed two times with phosphate buffered saline (PBS) and fixed by 4% paraformaldehyde for 20 min at 37 °C. After three times washing with PBS, nuclei were stained by 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI, 0.2 µg/mL, dissolved in PBS, Sigma-Aldrich Kft.) for 15 min. After washing, cover glasses were mounted to microscopy slides (VWR International, Debrecen, Hungary) by Mowiol® 4-88 mounting medium (Sigma-Aldrich Kft.). In case of the lysosomal co-localisation study, lysosomes were stained in living cells before treatment with peptide K2 by CytoPainter Lysosomal Staining Kit - Deep Red Fluorescence (abcam, Cambridge, UK), according to the manufacturer’s instructions. Confocal microscopy was carried out using a Zeiss LSM 710 system (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 40× oil objective. Images were processed by software ZEN Lite (Carl Zeiss Microscopy GmbH).
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