gb-MSCs (5,000 cells/well) were cultured in an eight-well chamber slide (Ibidi GmbH) at 37°C. Once cells reached 70% confluence, they were washed with PBS three times, fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton-X-100 for 20 min at room temperature and blocked with a solution containing donkey serum (Antgene) for 1 h at room temperature. Subsequently, cells were incubated with primary antibodies against PDGFR and TGFβ1-R (R&D Systems, Inc.) overnight at 4°C. After being washed three times with PBS, cells were incubated with secondary antibodies labeled with rhodamine (Antgene) for 1 h at room temperature. In each experiment, non-specific staining by secondary antibodies was excluded by incubating a well without primary antibodies. Cells were also stained with DAPI for 5 min at room temperature (Antgene). Images were acquired using a fluorescent microscope (Olympus Corporation) and data were analyzed using Image-Pro Plus v6.0 (Media Cybernetics, Inc.).
Donkey serum
Manufactured by Antgene
Sourced in China
Donkey serum is a biological product derived from the blood of donkeys. It is a complex mixture of proteins, hormones, and other biomolecules. The core function of donkey serum is to provide a source of these components for various research and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Eight well chamber slide, by Ibidi (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Antgene (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Desmin, by R&D Systems (1 mentions)
α sma, by R&D Systems (1 mentions)
Cd151, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Antgene (1 mentions)
Image pro plus, by Olympus (1 mentions)
2 protocols using donkey serum
1
Immunostaining of PDGFR and TGFβ1-R in gb-MSCs
2
Characterization of Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Gb-MSCs and bm-MSCs were grown on an eight-well chamber slide (Ibidi, Germany) until 70% confluent and washed with PBS three times. Then, the medium was replaced with serum-free medium (0% DMEM) and serum-free glioblastoma-conditioned medium (0% gb-CM), and the cells were incubated for 72 hours at 37°C. The cells were fixed with 4% paraformaldehyde, permeabilized with Triton-X-100 and blocked with solution containing donkey serum (Antgene, WuHan; China) for 1 h at room temperature. Then, the cells were incubated with primary antibodies against α-SMA, Desmin (RD, USA), NG2, Nestin, CD31, CD151, VE-cadherin (VE-cad) or smooth muscle myosin (SMM) (Abcam, USA) overnight at 4°C. After being washed with PBS, the cells were incubated with secondary antibodies labeled with either fluorescein or rhodamine (Antgene, WuHan; China) for 1 h at room temperature. In each experiment, nonspecific staining by secondary antibodies was excluded by incubating a well without primary antibodies. The staining was visualized using 4', 6-diamidino-2-phenylindole (DAPI) (Antgene, WuHan; China). A digital image was acquired using an Olympus (Olympus, Japan) camera, and the results were analyzed using Image-Pro Plus.
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