The third-passage cells were seeded at a density of 1 × 104/ml in a confocal dish (Nest). After the cells were completely attached on the next day, they were fixed with 4% paraformaldehyde for 10 min and blocked with 1% bovine serum albumin (BSA, Sigma) for 30 min at room temperature. Then the cells were incubated for 45 min at 4°C in the dark with phycoerythrin (PE)-conjugated anti-human CD29 monoclonal antibody (clone: TS2/16, BioLegend BD, ref. 303003; Table S1) and fluorescein isothiocyanate (FITC)-conjugated anti-sheep CD44 antibody (clone: 25.32, AbD Serotec, ref. MCA2219F; Table S1). The corresponding isotype control IgG was simultaneously incubated as a negative control (Table S1). Finally, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml; Solarbio) for 15 min. The fluorescently labeled cells were visualized and photographed using an inverted fluorescence microscope (Nikon Ti-S).
Confocal dish
Manufactured by NEST Biotechnology
Sourced in China
The Confocal dish is a specialized laboratory equipment designed for high-resolution imaging of cells and tissues. The dish features a transparent glass or plastic bottom that allows for the efficient transmission of light, enabling detailed microscopic examination of samples.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8, by Leica (2 mentions)
Triton x 100, by Solarbio (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Confocal laser scanning microscopy, by Olympus (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Actin tracker green, by Beyotime (1 mentions)
Actin tracker red, by Beyotime (1 mentions)
Ab150079, by Abcam (1 mentions)
Ab76149, by Abcam (1 mentions)
Confocal laser scanning microscopy, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
Accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lsm 900, by Zeiss (1 mentions)
Image ittm fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor plus 594 goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
29 protocols using confocal dish
1
Immunofluorescence Staining of CD29 and CD44
2
Immunofluorescence Imaging of Transfected Cells
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DF-1 cells in a confocal dish (Nest, China) were transfected with indicated plasmids (4 μg) for 24 h. The cells were then fixed with 4% paraformaldehyde for 10 min and penetrated with 0.2% Triton X-100 for 5 min at room temperature. The fixed cells were subsequently incubated with rabbit anti-Myc polyclonal antibodies for 2 h at room temperature. Next, the cells were stained with FITC-labeled goat anti-rabbit IgG for another 1 h at 37°C. The nucleus was then stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI ) (Roche, 10236276001). Fluorescence signals were scanned using an Olympus laser scanning confocal microscopy (Olympus Corporation, Tokyo, Japan).
3
Cellular Uptake of Engineered Extracellular Vesicles
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Naïve T cells and activated T cells which stimulated with anti‐CD3 and anti‐CD28 antibodies for 72 h were seeded in 24‐well plates. T cells were then incubated with MVs‐NPs, IFN‐γ‐MVs‐NPs, or FIRN labeled with Dil. After 1 h, 3 h, and 6 h of incubation, cells were collected and washed three times with cold PBS to remove free Dil. Cells were then stained with 4',6‐diamidino‐2‐phenylindole (DAPI) for 5 min. The cell sediment was suspended in 500 µL agarose gel (1%, W/V) and subsequently seeded in confocal dish (NEST). Images were obtained by laser scanning confocal microscope (Olympus FluoView FV 1000, USA).
4
Visualizing Actin Cytoskeleton Dynamics with LIPUS
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F-actin cytoskeletal staining was performed using the fluorescent dye Actin-Tracker Green (Beyotime, China). HUVECs were seeded on confocal dish (NEST, China) at a density of 2 × 104 cells/well. Subsequently, BTO nanoparticles were added for incubation, during which LIPUS treatment (1 min) was performed twice. After culturing for 48 h, the cells were washed with PBS, and then fixed with 4% paraformaldehyde (Biosharp, China) for 10 min. After washed with PBS containing 0.1% Triton X-100 (Sigma Aldrich, USA), the samples were sequentially stained with Actin-Tracker Green (1:200 in PBS solution, 1 h) solution and 4′,6-diamidino-2-phenylindole (3min; DAPI, Beyotime, China) solution for F-actin cytoskeleton and cell nucleus visualization respectively. Then the stained samples were observed by a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany).
5
Cellular Uptake of U-BTO/P-BTO in HUVECs
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HUVECs were seeded on a confocal dish (NEST, China) at a density of 2 × 104 cells/well and incubated for 12 h at 37 °C. Then, the medium was replaced with fresh ECM containing fluorescein isothiocyanate (FITC)-labeled U-BTO/P-BTO, after which cells were cultured for 0, 3, and 6 h, respectively. The cells were stained with Actin-Tracker Red (30 min, Beyotime, China) and DAPI (3min) at 37 °C in the dark. Fluorescent images were captured using CLSM.
6
Immunofluorescence Imaging of Cellular Proteins
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For immunofluorescence analysis, monolayers of cells were grown on a confocal dish (NEST, Guangzhou, China). The cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with PBS containing .5% TritonX‐100. Blocking was performed with 5% BSA, and the cells were subsequently incubated overnight at 4°C with primary antibody (1:100, ab76149, abcam). FITC‐conjugated rabbit anti‐goat IgG (1:500, ab150079, abcam) was used as the secondary antibody. The cell nuclei were counterstained with 10 μg/mL 4′,6‑Diamidino‑2‑phenylindole (DAPI) solution (C0065, Solarbio, Beijing, China). Images were obtained and analyzed using a confocal microscope (TCS‐SP8, Leica, Wetzlar, Germany).
7
Quantitative Biofilm Analysis Protocol
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We diluted the obtained fresh bacteria liquid 100-fold with TSBG (TSB with 0.25% glucose) and the diluted bacteria were added in a confocal dish (diameter 15 mm, Nest) mixed with GNCs-CBM hydrogel. After incubation at 37 °C for 24 h, biofilm formation was determined by crystal violet staining. After discarding the medium, PBS was slightly added to wash away the planktonic bacteria. Biofilms were dried for 20min in the air after being fixed in methanol. Then, the biofilms were stained with 0.5% crystal violet. The stain dye was removed after 20 min and each dish was washed twice with PBS. The stained biofilms were re-solubilized in 33% ethanoic acid and measured at OD550 using the microplate reader.
8
Immunofluorescence Staining of Cells
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Cells were seed into confocal dish (NEST) and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. After permeabilization with 0.5% Triton X-100 for 10 min, cells were blocked with 5% goat serum and incubated with primary antibody overnight at 4 °C. The next day, cells were washed and incubated with secondary antibody at room temperature for 1 h, and then were labeled the nuclei by using Hoechst 33342 for 5 min.
9
Visualizing Podocyte Cytoskeleton Dynamics
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Podocytes were plated on a confocal dish (NEST, UK) and treated with relevant treatment for 24 h. After treatment, the podocytes were washed three times with PBS and fixed with 4% paraformaldehyde, followed by permeabilization and blocking with 0.3% Triton X-100 and 10% goat serum. Alexa-phalloidin (Invitrogen, USA) was used to stain F-actin. After three washes with PBS, the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA). Stained images were obtained by confocal laser-scanning microscopy (Zeiss, Germany).
10
Cytotoxicity Assay for SMMC-7721 and Huh7 Cells
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SMMC-7721 and Huh7 cells (8 × 104) were seeded in a confocal dish (NEST Biotechnology). DSF/Cu (0–1 μM) was added into the indicated wells for 12 h. Then, the cells were incubated with Calcein-AM and PI for the staining of live and dead cells. Nuclei were also counterstained with Hoechst 33342 (1 μg/mL) for 10 min. The fluorescence was observed under a confocal microscope (Leica TCS SP5, German).
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