By using EdU (5-ethynyl-20-deoxyuridine) analysis resulting from Cell-Light EdU DNA Cell Proliferation Kit, we surveyed the proliferation of cells. Cultured at 5% CO2 with a temperature of 37°C for 48 h, the 1 × 104 cells were seeded into plates with 96 wells. Every well with cells cultivated for as long as 2 h was added with Edu solution with a 50 μM of eventual concentration. After that, cells were fixed by using 4% paraformaldehyde and dyed by applying Apollo dye solution for proliferation exploration. By using a fluorescence microscope, the observation, calculation, and photographing of the cells with positive EdU were carried out after we washed the cells with PBS (Olympus, Tokyo, Japan).
Phosphate buffered saline (pbs)
Manufactured by Olympus
Sourced in Japan
PBS (Phosphate-Buffered Saline) is a commonly used buffer solution in various laboratory applications. It is an aqueous solution containing sodium chloride, sodium phosphate, and potassium phosphate. The primary function of PBS is to maintain a stable pH and isotonic environment for biological samples, cells, or reagents.
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Lab products found in correlation
Fluorescence microscope, by Olympus (1 mentions)
Alexa fluor 488 conjugated goat anti rat igg, by Abcam (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Freezing microtome, by Leica camera (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
Phosphate buffered saline (pbs), by Beyotime (1 mentions)
Vs200 research slide scanner, by Olympus (1 mentions)
Phosphate buffered saline (pbs), by ZSGB-BIO (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Toluidine blue, by Leagene (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Optical microscope, by Olympus (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Imt 2, by Olympus (1 mentions)
Evans blue solution, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
6 protocols using phosphate buffered saline (pbs)
1
Quantifying Cell Proliferation with EdU
2
Immunocytochemistry of C2C12 Myotubes
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C2C12 myotubes that seeded onto glass coverslips in six‐well culture plate were fixed with 4% paraformaldehyde in PBS (Genom Biotech Ltd., Hangzhou, China) for 30 min and stained with Hoechst 333258 (Beyotime, China) for 20 min at room temperature. Then, cells were washed with PBS three times and examined under a fluorescence microscope (Olympus, Tokyo, Japan).
3
Quantifying IgG Extravasation in Rat Brain
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BBB disruption could be also assessed through a one-step immunohistochemical detection of IgG24 (link). The rats were transcardially perfused with PBS (pH 7.2 to 7.4, Beyotime) followed by ice-cold 4% paraformaldehyde (Servicebio, Wuhan, China). Then, rat brains were removed, post-fixed in 4% paraformaldehyde (Servicebio), cryoprotected in 15% and 30% sucrose in PBS (pH 7.2 to 7.4) (Beyotime) in turn for 2 days, and then sliced into 20 mm frozen sections on a freezing microtome (Leica, Japan). The sections were rinsed thrice with PBS (pH 7.2 to 7.4, Beyotime), followed by blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h, and then the sections were incubated 1 h at room temperature with Alexa Fluor 488® conjugated goat anti-rat IgG (Abcam). After three rinses with PBS (pH 7.2 to 7.4, Beyotime) for 5 min each time, the fluorescent images were visualized with a VS200 Research Slide Scanner (Olympus, Tokyo, Japan), and the fluorescence intensity of lgG and area of lgG extravasation were measured by Image J software.
4
Cell Fixation and Staining Protocol
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Cells were digested with 0.2% trypsin (Beyotime Biotech, Shanghai, China), re-suspended with DMEM (Gibco BRL. Co.), and the cell density was adjusted to 2×104/ml. In 12-well plates, the cell climbing pieces (round pieces) were placed. The cell suspension was dropped onto the pieces and cultured for 30 min. Then, DMEM containing 10% FBS was added to 12-well plates and cultured at 37°C and 5% CO2 in an incubator for 24 h. The pieces were washed using PBS (ZSGB-Bio., Beijing, China) 3 times (5 min per time) and incubated with 4% paraformaldehyde (Beyotime Biotech) at room temperature for 20 min. Subsequently, the cells on pieces were stained using toluidine blue (Leagene Biotech. Co., Beijing, China) for 5 min and rinsed with PBS for 30 s. Finally, the pieces were air-dried naturally and sealed with neutral gum and observed under microscopy (Mode: BX51, Olympus, Tokyo, Japan).
5
Quantitative Analysis of Osteogenic Differentiation
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We seeded hUCMSCs in 6-well plates at a density of 200,000 cells per well and cultured them in osteogenic medium. We replaced the medium every two days. On day 7, ALP staining was performed using a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) following the instructions from the manufacturer [31 (link)]. Briefly, we fixed the cells in 4% paraformaldehyde (Solarbio, China) for 30 min at room temperature (RT), washed the cells three times with PBS (GIBCO, USA), and stained the cells with NBT/BCIP solution for 24 h at RT. Then we removed the staining solution, washed the cells three times with PBS, and observed them under an optical microscope (OLYMPUS, Japan). The ALP staining images were semi-quantified using ImageJ (version 1.6.0, Wayne Rasband, National Institute of Health, USA) as described previously [32 (link),33 ].
6
Rabies Virus Antigen Detection Assay
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Cells were cultured in 96-well plates for 24 h and then inoculated with samples diluted in culture medium for 2 h. Sample media were replaced by fresh medium and cells were incubated for 48–92 h. After that, the medium was completely removed, and cells were fixed with cold acetone 80% in ice bath for 15 min and the plates were allowed to dry in a safety cabinet for 10 min. Cells were then stained with fluorescein isothiocyanate (FITC)-labeled anti-rabies antibody (BioRad, FR) diluted 1/20 in 1/40.000 Evans Blue solution in PBS (Sigma-Aldrich, US) for 30 min at 37°C. Plates were washed twice with PBS and cells were examined using a fluorescence microscope IMT-2 (Olympus, JP). All wells were fully screened at 40x magnification and cells containing small fluorescent cytoplasmic granules were identified as antigen-positive after confirmation at 200x magnification. Positive and negative controls for DFA were done in each plate, where cells were incubated with WRV or simply media for controls in 2 wells.
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