Immunofluorescence analysis was performed on cell-paved µ-slides. The abovementioned cells were fixed with ice-cold 4% paraformaldehyde for 20 min at 37°C, blocked with normal serum for 20 min at room temperature and then incubated overnight in the dark at 4°C with specific antibodies against mouse monoclonal CD34 (ab 8158; Abcam, Cambridge, UK) and mouse monoclonal factor VIII (9035; Invitrogen™; Thermo Fisher Scientific, Inc.). After three washes, the slides were stained with Cy3-conjugated anti-mouse antibodies or anti-rabbit antibodies (1:200; Abcam). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and the stained sections were then visualized with a confocal microscope (Olympus Corp., Tokyo, Japan).
Anti rabbit antibodies
Manufactured by Abcam
Sourced in United Kingdom
Anti-rabbit antibodies are immunoglobulin molecules that recognize and bind to rabbit proteins. They are used in various laboratory techniques, such as Western blotting, immunohistochemistry, and ELISA, to detect and quantify rabbit-derived proteins or to isolate and purify rabbit-specific targets.
Automatically generated - may contain errors
Lab products found in correlation
Ab8158, by Abcam (1 mentions)
Cy3 conjugated anti mouse antibodies, by Abcam (1 mentions)
Alphaeasefc, by Bio-Techne (1 mentions)
V300 scanner, by Epson (1 mentions)
Electrogenerated chemiluminescence, by Cytiva (1 mentions)
Ab23981, by Abcam (1 mentions)
Ab75285, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab43791, by Abcam (1 mentions)
Ab93876, by Abcam (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
4 protocols using anti rabbit antibodies
1
Immunofluorescence Analysis of Cell Markers
2
Apoptotic Protein Expression Analysis
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Caspase-3 is an enzyme that is one of the most important enzymes of the process of apoptosis. It is considered that BAX is one of the most important apoptosis genes which belong to the Bcl-2 gene family. And the ratio of BAX/Bcl-2 is a key factor of determining the inhibition of apoptosis. Moreover, cytochrome c is also involved in initiation of apoptosis. Therefore, immunohistochemical examination and western blot analysis were used to detect the levels of caspase-3, BAX, Bcl-2 and cytochrome c. All of the secondary anti-rabbit antibodies were purchased from Abcam, UK. Frozen heart, liver and kidney tissues were homogenized by following the manufacturer's instruction. Whole protein extract was used to detect the expression of apoptotic protein. After immunoblotting, the blot was scanned by EPSON V300 Scanner. Alpha Innotech alphaEaseFC was performed to analyze optical density. And GAPDH was used as normalization of intensity of immunoblot bands.32 (link)
3
CD44 Immunofluorescence Staining Protocol
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The cell density was adjusted to 3×106/ml and the cells were laid in the glass bottom dishes specially designed for laser confocal microscopy. Cells were fixed with 4% paraformaldehyde for 20 min at 4°C and permeabilized with 0.1% Triton X-100 for 10 min, blocked with 3% BSA, and sealed for 60 min at room temperature. After incubation overnight with anti-CD44 antibody (1:200; cat. no. ab189524; Abcam), the cells were rinsed thoroughly and treated with anti-rabbit antibodies (1:200; cat. no. BA1032; Boster Biological Technology), respectively. Nuclei were stained using 0.3 µM DAPI (cat. no. C1002; Beyotime Institute of Biotechnology).
4
Western Blot Analysis of Bone Marrow Stromal Cell Proteins
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Whole-cell lysates were prepared from the BMSCs. Cells were lysed using mammalian protein extraction reagent (Pierce Chemical, Dallas, TX), containing protease inhibitor mixture (Roche Applied Science, Indianapolis, IN). The whole-cell lysates (10 μg protein/lane) were loaded and separated in 10% sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) gels, electro-blotted into a nitrocellulose membrane, and immune-blotted with anti-rabbit antibodies (Abcam, Cambridge, UK) to GAPDH (ab181602, 1:10,000), KLF2 (ab139699, 1:1000), runt-related transcription factor 2 (Runx2, ab23981, 1:1000), osteocalcin (OCN, ab93876, 1:500), osteopontin (OPN, ab75285, 1:1000), WWP1 (ab43791, 1:1000), CD63 (ab134045, 1:1000), CD81 (ab109201, 1:1000), Calnexin (ab92573, 1:20,000), TSG101 (ab125011, 1:1000), p65 (ab32536, 1:1000), phosphorylated p65 (ab86299, 1:2000), IκBα (ab32518, 1:1000), and phosphorylated IκBα (ab133462, 1:10,000). The electrogenerated chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) was employed to visualize these bands.
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