Coumarin 6 was utilized to visualize encapsulation into the shell of PBCA MB. A Leica TCS SP8 X inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a plan-apochromat 100x/1.40 oil-immersion objective was used to visualize coumarin 6-loading in PBCA MB. The samples were applied on a high precision cover glass (170 μm, No. 1.5H) from Marienfeld (Lauda-Königshofen, Germany). The excitation wavelength was set at λ = 470 nm via a filtered white light laser and the resulting emission was detected at λ = 491 - 556 nm. Deconvolution of the images was processed using the Huygens Professional software via the Classic Maximum Likelihood Estimation (CMLE) method.
High precision cover glass
Manufactured by Marienfeld
Sourced in Germany
High precision cover glass is a thin, transparent glass slide used to cover and protect microscope samples. It provides a flat, even surface to ensure optimal light transmission and image clarity during microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Leica tcs sp8 x inverted, by Leica Microsystems (1 mentions)
No 1.5h, by Marienfeld (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Em grade formaldehyde, by Polysciences (1 mentions)
Mowiol, by Thermo Fisher Scientific (1 mentions)
Doxycycline dox, by Merck Group (1 mentions)
2 protocols using high precision cover glass
1
Coumarin 6 Visualization of PBCA Microbubbles
2
Immunofluorescence Imaging of Cell Lines
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Cell cultures. Cell lines were cultured as recommended by ATCC guidelines. For live-cell imaging, cells were seeded into glass bottom imaging dishes (Lab-Tek, Nunc; MatTek, MatTek Corporation).
For immunofluorescence studies, cells were grown on high Precision cover glass (Marienfeld). For confocal microscopy, cells were fixed with 4% EM grade Formaldehyde (Polysciences) for 5 minutes at 37 C. Cells were then permeabilized by treatment with 0.16% Triton X-100 for 2 minutes. Primary and secondary antibodies were diluted in PBS/0.05% Saponin and incubated for 1 to 2 hours. After antibody staining, samples were mounted on microscope slides (Menzel-Glaser) with Mowiol for standard confocal imaging or SlowFade Gold Antifade Reagent (Life Technologies) for Airyscan microscopy. RPE1 and HeLaK cell lines stably expressing inducible CHMP7 or LEMD2 alleles were treated with 500 ng/ml doxycycline (DOX, Sigma-Aldrich). XPO1 inhibition was carried on by incubating cells with 2.5 ng/ml Leptomycin B (LMB, InvivoGen) for up to 3 hours. Loss of XPO1 activity was monitored my nuclear localization of 2xmRuby3-NES. Micronucleation was induced by MPS1 inhibitor AZ3146 (Selleckcem) used at a concentration of 2 µM. RPE1 cell lines were depleted for p53 before AZ3146 treatment.
For immunofluorescence studies, cells were grown on high Precision cover glass (Marienfeld). For confocal microscopy, cells were fixed with 4% EM grade Formaldehyde (Polysciences) for 5 minutes at 37 C. Cells were then permeabilized by treatment with 0.16% Triton X-100 for 2 minutes. Primary and secondary antibodies were diluted in PBS/0.05% Saponin and incubated for 1 to 2 hours. After antibody staining, samples were mounted on microscope slides (Menzel-Glaser) with Mowiol for standard confocal imaging or SlowFade Gold Antifade Reagent (Life Technologies) for Airyscan microscopy. RPE1 and HeLaK cell lines stably expressing inducible CHMP7 or LEMD2 alleles were treated with 500 ng/ml doxycycline (DOX, Sigma-Aldrich). XPO1 inhibition was carried on by incubating cells with 2.5 ng/ml Leptomycin B (LMB, InvivoGen) for up to 3 hours. Loss of XPO1 activity was monitored my nuclear localization of 2xmRuby3-NES. Micronucleation was induced by MPS1 inhibitor AZ3146 (Selleckcem) used at a concentration of 2 µM. RPE1 cell lines were depleted for p53 before AZ3146 treatment.
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