Immunofluorescence staining of cells was performed in chambered slides (BD Bioscience, San Jose, CA, USA). Cells were seeded in chambered slides and fixed in ice-cold methanol for 10 min. Slides were then washed in phosphate-buffered saline (PBS) three times, and they were incubated with blocking solution (3% BSA in PBS) for 30 min. For immunofluorescence staining of mouse tissues, formalin-fixed, paraffin-embedded sections were used. Slides were deparaffinized and dehydrated. Mouse monoclonal acetylated α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) and non-phospho β-catenin antibody (Cell signaling, Danvers, MA, USA) was used as primary antibodies. Samples were then stained with Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG antibody (Life technologies, Carlsbad, CA, USA) and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, CA, USA) with DAPI (4′,6-diamidino-2-phenylindole).
Chambered slides
Manufactured by BD
Sourced in United States
Chambered slides are specialized microscope slides that feature one or more separate compartments, or chambers, within the slide. These chambers allow for the containment and observation of small samples or specimens, such as cells, tissues, or liquids, under a microscope. Chambered slides provide a controlled environment for the samples and facilitate various microscopy techniques.
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Lab products found in correlation
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Non phospho β catenin antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 568 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Recombinant wnt3a, by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Six well plate, by Greiner (1 mentions)
Polylysine, by Merck Group (1 mentions)
D9542, by Merck Group (1 mentions)
Orca r2 digital ccd camera c10600, by Hamamatsu Photonics (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Goat anti sox17, by R&D Systems (1 mentions)
Mu392a uc, by Abcam (1 mentions)
Rabbit anti trp53, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using chambered slides
1
Immunofluorescence Staining of Cells and Tissues
2
Regulating Ciliogenesis in Proximal Tubular Cells
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HK2 human proximal tubular epithelial cells were cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 100 g/mL penicillin, 100 g/mL streptomycin, and 10% fetal bovine serum (FBS, Cellgro, Manassas, VA, USA) at 37°C in 5% CO2. For immunostaining of cilia in culture, cells are grown as a monolayer on chambered slides (BD Bioscience, San Jose, CA, USA) and for RNA isolation in six-well plates (Greiner Bio-One, Kremsmunster, Austria). To generate ciliated and non-ciliated tubular epithelial cells, we seeded HK2 cells at different densities (six-well plates; 5,000 vs 30,000 per well in six-well plates) and starved cells for 48 h without serum to induce cell cycle exit and ciliogenesis as described previously [38 (link)]. Upon serum starvation, we exchanged media with growth factor-free control media or with media supplemented with 100 ng/ml recombinant Wnt3a (R&D systems, Minneapolis, MN, USA) for 48 h.
3
Smad3-mediated VEGF-A regulation in vascular SMCs
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Vascular SMCs were seeded at 50–60% confluence in DMEM medium in chambered slides (BD Biosciences) and incubated for 24 h. The cells were then infected with AdSmad3 or AdGFP and then treated with recombinant TGF-β (5 ng/ml) or solvent for 72 h. Conditioned media from AdGFP-treated or AdSmad3/TGF-β-treated SMCs were collected and incubated with 10 μg/ml VEGF-A-neutralizing antibody (Nab) or normal goat IgG for 1 h. The antibody-treated conditioned media were then added to naive SMCs and incubated for 30 min. Cells were fixed in 4% paraformaldehyde at room temperature for 10 min and permeabilized with 0.2% Triton X-100. Immunostaining for injured rat carotid arteries with or without Smad3 overexpression was performed as described previously.
4
Imaging Localization of T. brucei
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Tissue culture glass slides with 8 chambers were treated with 0.1% Poly-Lysine (Sigma, P-8920). 2.5 × 106 formaldehyde-fixed T. brucei were allowed to adhere to Poly-Lysine-treated chambered slides (Falcon, 354108), permeabilised with 0.2% (w/v) Triton X-100 then incubated with protein-specific antibodies followed by fluorescently-labelled second antibodies in PBS containing 0.5% gelatin. DNA was stained with 100 ng/ml DAPI (D9542, Sigma-Aldrich). Mitochondria were detected by addition of Mitotracker Red CMXRos (50 nM, Thermo Fisher Scientific) to the cells 5 min prior to fixation. Images were examined using the Olympus IX81 microscope, 100x Oil objective with a numerical aperture of 1.45. Digital images were taken with ORCA-R2 digital CCD camera C10600 (Hamamatsu) and using the Xcellence RT software. The bright-field images were taken using differential interference contrast (DIC). Fluorescent images were taken as Z-Stacks with a height of roughly 4 µm and a step width of 0.2 µm. The images were deconvoluted (Wiener Filter, Sub-Volume overlap: 20) and then processed using ImageJ. The background was subtracted and brightness and contrast were adjusted automatically. The most in-focus image of the deconvoluted stack was used.
5
Immunofluorescence of Mouse Blastocysts
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