Sterile glass coverslips were placed in each well of a 24-well tissue culture plate, and 7.5 × 104 J774A.1 cells were added to each well in DMEM medium 24 h prior to infection. Cells were then infected with L. pneumophila from a 48 h heavy patch with an estimated MOI of 20 and infections were synchronized by spinning plates at 200 × g for 5 min, followed by an incubation at 37 °C. 1, 4, and 8 h after infection cells were fixed with 4% PFA for 10 min. L. pneumophila cells were stained using a rabbit anti-Legionella antibody49 (link) (1:1000) followed by an Alexa Fluor 488-conjugated secondary antibody (Invitrogen, 1∶2000) and then washed extensively with PBS. 4,6-diamidino-2-phenylindole staining (DAPI, 0.1 μg ml−1, Life Technologies) was used to identify host cell nuclei and bacterial cells. In all experiments coverslips were mounted using the ProLong gold antifade reagent (Invitrogen). Coverslips were imaged on a Nikon Eclipse TE2000-S inverted fluorescence microscope with a ×100/1.4 numerical aperture objective lens. The microscope camera was a Photometrics CoolSNAP EZ camera controlled by SlideBook™ (Intelligent Imaging Innovations).
4 6 diamidino 2 phenylindole staining dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
4,6-diamidino-2-phenylindole (DAPI) is a fluorescent stain that binds to DNA. It is commonly used in microscopy and fluorescence-based assays to visualize and identify cell nuclei.
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Lab products found in correlation
Eclipse te2000 s, by Nikon (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Glutamax 1 100x, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum superior, by Merck Group (1 mentions)
Pkh26 dye, by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Tissue tek oct compound, by Bio-Optica (1 mentions)
Anti parvalbumin, by Swant (1 mentions)
Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Polysine microscope adhesion slides, by Thermo Fisher Scientific (1 mentions)
4 protocols using 4 6 diamidino 2 phenylindole staining dapi
1
Quantifying Legionella Pneumophila Infection In Macrophages
2
Cell culture of meningioma cell lines
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The benign cell line Ben-Men-1 (Leibniz Institute DSMZ, Braunschweig, Germany), the anaplastic meningioma cell line NCH93 [10 (link)] and IOMM-Lee were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies Limited, Paisley, UK) supplemented with 10% fetal bovine serum superior (Sigma-Aldrich, St. Louis, MO, USA), 2% GlutaMAXTM-I (100X, Life Technologies Corporation, Grand Island, NY, USA), and 1% Penicillin/Streptomycin (Life Technologies Corporation, Grand Island, NY, USA) at 37 °C in a humidified environment with 5% CO2 atmosphere. Mycoplasma contamination was excluded by 4′,6-diamidino-2-phenylindole staining (DAPI, Life Technologies Corporation, Eugene, OR, USA). NCH93 cell line was authenticated by STR DNA profiling analysis (Leibniz Institute DSMZ, Braunschweig, Germany).
3
Internalization of mEVs in Caco-2 Cells
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B-mEVs and H-mEVs (100 μg) were labeled with 100 μl of the 40 μM protein-binding dyes CFSE [5-(and-6)-carboxyfluorescein diacetate succinimidyl ester; Thermo Fisher Scientific, USA] by incubation at 37°C for 2 hours. Free dye was removed by ultrafiltration at 2000g for 30 min. Labeled CFSE-mEVs were incubated with Caco-2 cells at 37°C for 8 hours, and then the cells were harvested and washed to remove free particles. For confocal microscopy, samples were fixed with 4% paraformaldehyde and washed twice with PBS. Afterward, PKH26 dyes (Sigma-Aldrich, MINI26-1KT, USA) were used to stain cell membrane, and LysoTracker Deep Red (Invitrogen, L12492, USA) were used to stain lysosomes in Caco-2 cells. Cell nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (Thermo Fisher Scientific, 62248, USA).
4
Immunohistochemistry of Brain Tissue
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Mice were anesthetized and perfused transcardially with sucrose 5% and paraformaldehyde 4%. Brains were removed and post-fixed overnight in paraformaldehyde 4%. The day after three washes with PBS were performed and brains were incubated for at least eight hours in 30% sucrose. Finally, brains were included in cryomolds with Tissue-Tek OCT compound (Bio-Optica) and put at − 80 °C until cryostat sectioning. Brains were cut with a cryostat and 20 μm-thick coronal slices were collected on polysine microscope adhesion slides (ThermoFisher). Slices were incubated first in blocking solution (3%BSA, 10% goat serum, 0.4% Triton-X-100, diluted in PBS) for at least 30 min and then they were incubated with primary antibodies overnight at 4 °C. Subsequently, three wash (10 min each) with PBS were performed and brain slices were incubated with fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After the antibody incubation, brain slices were washed and 4',6diamidino-2-phenylindole (DAPI) staining (ThermoFisher) was carried out (DAPI diluted in PBS to a final concentration of 0.5 μg/ml). Finally, another washing step is performed before mounting the coverslips with Mounting Medium (Vecta Shield). Primary antibodies used were: anti-Parvalbumin (Swant, GP72), Shank3 (homemade SHANK3rb [35 (link)]).
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