BMSCs were fixed with 4% paraformaldehyde after 7 days of growth on the CPC surface. Cells were permeabilized with 0.5% Triton X-100 for 10 min. Samples were washed thrice with PBS for 5 min and incubated with 3% bovine serum albumin solution for 30 min, followed by incubating with primary mouse anti-osteopontin (OC, 1:500, Abcam, UK) and rabbit anti-RUNX2 (1:500, Affinity, China) antibodies overnight at 4 °C. After washing thrice for 5 min each in PBS, Alexa Fluor 594-labeled donkey anti-mouse and Alexa Fluor 488-labeled donkey anti-rabbit (1:1000, Abcam, UK) were mixed and incubated for 2 h at 37 °C in dark. Samples were then washed with PBS and nuclei were stained with 10 ng/mL DAPI (4′,6-diamidino-2-phenylindole, Beyotime, China). Finally, the samples were imaged by a confocal laser scanning microscope (Zeiss LSM 800, Germany).
Alexa fluor 594 labeled donkey anti mouse
Manufactured by Abcam
Sourced in United Kingdom
Alexa Fluor 594-labeled donkey anti-mouse is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize mouse primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using alexa fluor 594 labeled donkey anti mouse
1
Immunostaining of Osteogenic Markers
2
Bone Cement Interaction with Cells
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Cells were cultured together with bone cement material for 14 days, the medium was removed, and cells were washed lightly with PBS 3 times. Cells were fixed with 4% paraformaldehyde for 10 min and then soaked with 0.1% Triton X-100 for 15 min to make the cell membrane permeable. Subsequently, cells were incubated with primary mouse anti-alkaline phosphatase (ALP, 1:500, Abcam, UK) or mouse anti-osteopontin (OPN, 1:500, Abcam; Cambridge, UK) for 12 h at 4 °C after blocking with bovine serum albumin (BSA) for 1 h. After washing 3 times for 5 min each in PBS, Alexa Fluor 594-labeled donkey anti-mouse (1:1000, Abcam; Cambridge, UK) was incubated for 2 h at 37 °C in the dark. Cells were then washed with PBS and incubated with DAPI (40,6-diamidino-2-phenylindole, Beyotime; Shanghai, China) solution and phalloidin (Solarbio; Beijing, China) for 30 min. After washing with PBS, the stained cells were observed under the microscope (Leica; Weztlar, Germany).
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