For the confocal imaging of 293T cells, high precision microscope cover glasses (Marienfeld) were coated with poly-L-lysine hydrobromide (p6282, Sigma-Aldrich) according to the manufacturer’s protocol. Cells were seeded onto cover glasses in normal growth medium and fixed in 4% Formaldehyde solution (AppliChem) in PBS 1x after 24h of incubation. Permeabilization and blocking of samples was performed in blocking solution (10% FCS, 0.3% Saponin (47036, Sigma-Aldrich) in PBS 1x) for 1h rocking. Anti-V5 Tag primary antibody (Thermo Fischer Scientific, #46-0705) was diluted 1:500 in blocking solution and applied for 2h at room temperature, rocking. Samples were washed three times in blocking solution and anti-mouse Alexa Fluor 594 (Thermo Fischer Scientific, #A-11005) was applied 1:400 in blocking solution for 1h at room temperature, rocking. After three times washing in blocking solution nuclei were counterstained with DAPI 1:1000 in PBS 1x, for 10min, rocking. Cover glasses were mounted onto microscopy slides using ProLong Gold (Thermo Fischer Scientific) antifade mountant. Image acquisition was performed on a confocal laser scanning microscope (Zeiss LSM 780, Carl Zeiss AG), equipped with an Airyscan detector using ZEN black 2.3 (Carl Zeiss AG).
High precision microscope cover glasses
Manufactured by Marienfeld
High precision microscope cover glasses are thin, transparent glass plates used to cover and protect samples being observed under a microscope. They provide a flat, smooth surface that helps to maintain the focus and clarity of the microscopic image.
Automatically generated - may contain errors
Lab products found in correlation
Poly l lysine hydrobromide, by Merck Group (2 mentions)
Formaldehyde solution, by AppliChem (2 mentions)
Anti v5 tag primary antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Zen black 2, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Small cover glass, by Avantor (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Voltalef, by Avantor (1 mentions)
Onemp, by Refeyn (1 mentions)
5 protocols using high precision microscope cover glasses
1
Confocal Imaging of V5-tagged 293T Cells
2
Confocal Imaging of V5-tagged 293T Cells
3
Accessory Gland Tissue Preparation
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Accessory glands were prepared following the protocol described by (Fan et al., 2019) (link).
In brief, adult male flies were anaesthetised using CO2 and dissected in ice-cold 1× PBS (Gibco, Thermo Fisher Scientific). The whole male reproductive system was carefully pulled out of the body cavity, keeping testis, accessory gland, ejaculatory duct and ejaculatory bulb to avoid stress in the tissue. They were then stained with ice-cold 500 nM LysoTracker® Red DND-99 (Invitrogen®) in 1 × PBS for 5 min, washed in ice-cold 1 × PBS for 1 min, then mounted onto High Precision microscope cover glasses (thickness No. 1.5H, Marienfeld-Superior). A custom-built holder was used to hold the specimen during imaging. To avoid dehydration and hypoxia, the glands were maintained in a small drop of 1 × PBS, surrounded by 10S Voltalef® (VWR Chemicals), held by a small cover glass (VWR).
In brief, adult male flies were anaesthetised using CO2 and dissected in ice-cold 1× PBS (Gibco, Thermo Fisher Scientific). The whole male reproductive system was carefully pulled out of the body cavity, keeping testis, accessory gland, ejaculatory duct and ejaculatory bulb to avoid stress in the tissue. They were then stained with ice-cold 500 nM LysoTracker® Red DND-99 (Invitrogen®) in 1 × PBS for 5 min, washed in ice-cold 1 × PBS for 1 min, then mounted onto High Precision microscope cover glasses (thickness No. 1.5H, Marienfeld-Superior). A custom-built holder was used to hold the specimen during imaging. To avoid dehydration and hypoxia, the glands were maintained in a small drop of 1 × PBS, surrounded by 10S Voltalef® (VWR Chemicals), held by a small cover glass (VWR).
4
Mass Determination of Protein Samples
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Protein samples were diluted from 10x stocks to end concentrations of 20–100 nM in 50 mM Tris-buffer at pH 8.0 and 150 mM NaCl. The measurements were performed on a Refeyn OneMP using cleaned High Precision Microscope Cover Glasses (24 × 60 mm, Marienfeld) and silicon gaskets (CultureWellTM reusable gaskets, SKU: 103250). The gaskets were filled with 18 μL buffer to adjust the focus using the maximum sharpness level between 6 and 7. Then, 2 μL of the protein solution were added and briefly mixed before starting the acquisition of 6000–12000 images. To convert the obtained contrast values to molar masses, a standard curve was prepared prior to the experiment in the same buffer as described above using known masses from the NovexTM NativeMARKTM protein standard (Invitrogen). All solutions and buffers were sterile-filtered prior to use. The histograms were processed and fitted with the Refeyn DiscoverMP analysis software (v2.2.0).
5
Functionalized Microscope Cover Glass Protocol
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High-precision microscope cover glasses
(22 × 22 mm2, thickness 170 ± 5 μm; no.
1.5; from Marienfeld) were cleaned by sonication for 20 min in acetone.
For functionalization, slides were treated in a UV-ozone photoreactor
(Ultra Violet Products, PR-100) for 20 min to activate the surface
and were immediately functionalized with methacrylate groups by spin
coating (3000 rpm, 45 s) a 3-(trimethoxysilyl)propyl methacrylate
solution (1 vol % solution in a 1:1 water-isopropanol mixture) followed
by a curing step of 10 min at 100 °C. Following this, the functionalized
slide was attached to a second, unfunctionalized slide, using a 50
μm-thick double adhesive tape spacer, to form a simple cell
assembly.
(22 × 22 mm2, thickness 170 ± 5 μm; no.
1.5; from Marienfeld) were cleaned by sonication for 20 min in acetone.
For functionalization, slides were treated in a UV-ozone photoreactor
(Ultra Violet Products, PR-100) for 20 min to activate the surface
and were immediately functionalized with methacrylate groups by spin
coating (3000 rpm, 45 s) a 3-(trimethoxysilyl)propyl methacrylate
solution (1 vol % solution in a 1:1 water-isopropanol mixture) followed
by a curing step of 10 min at 100 °C. Following this, the functionalized
slide was attached to a second, unfunctionalized slide, using a 50
μm-thick double adhesive tape spacer, to form a simple cell
assembly.
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