Cell cultures were harvested after exposure to colcemid for 4 hours and chromosome preparations were obtained according to standard cytogenetic methods. However, for interphase analysis, samples were produced without colcemid treatment. Cells were spread onto slides and were then denatured in 70% formamide in 2× SSC at 70°C for 5 minutes. The probes were prepared using purified DNA from RP11-265B8 and RP11-876N18 BAC clones, which were labelled by nick translation with biotin and digoxigenin, respectively, according to the manufacturer’s instructions (Roche). The labelled DNAs were ethanol precipitated together with Cot-1 human DNA (Roche) and resuspended in 10 μl of hybridisation buffer (Sigma). The probes were incubated at 65°C for 10 minutes, followed by preannealing at 37°C for 10 minutes. Hybridisation was carried out at 37°C overnight followed by washing with 2× SSC for 5 minutes. The RP11-265B8 probe was detected with Avidin D-Texas Red, biotinylated anti-Avidin D and Avidin D-Texas Red (Vector Laboratories). The RP11-876N18 was detected with mouse anti-digoxigenin antibody (Sigma-Aldrich) followed by rabbit anti-mouse FITC and anti-rabbit FITC (Sigma-Aldrich). The slides were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) containing DAPI counterstain.
Rabbit anti mouse fitc
Manufactured by Merck Group
Sourced in Italy
Rabbit anti-mouse FITC is a fluorescently labeled secondary antibody that binds to mouse primary antibodies. It is a tool used in various immunoassay techniques, such as flow cytometry, immunofluorescence, and Western blotting, to detect and visualize the presence of mouse antigens.
Automatically generated - may contain errors
Lab products found in correlation
Hybridisation buffer, by Merck Group (2 mentions)
Cot 1 human dna, by Roche (2 mentions)
Texas red avidin d, by Vector Laboratories (2 mentions)
Mouse anti digoxigenin antibody, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Biotinylated anti avidin d, by Vector Laboratories (1 mentions)
3 protocols using rabbit anti mouse fitc
1
Interphase FISH Analysis of Genomic Regions
2
Fluorescence In Situ Hybridization Assay
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Cell cultures were harvested after exposure to Colcemid for 4 hours and chromosome preparations were obtained according to standard cytogenetic methods. For interphase analysis, samples were produced with the exception of Colcemid treatment. Cells were spread onto slides and were then denatured in 70% formamide in 2× SSC at 70°C for 5 minutes. The probes were prepared using purified DNA from RP11-265B8 and RP11-876N18 BAC clones, which were labeled by nick translation with biotin and digoxigenin respectively, according to the manufacturer's instructions (Roche, Mannheim, Germany). The labeled DNAs were ethanol precipitated together with Cot-1 human DNA (Roche) and resuspended in 10 µl of hybridisation buffer (Sigma). The probes were denatured by incubating at 65°C for 10 minutes, followed by preannealing at 37°C for 10 minutes. Hybridisation was at 37°C overnight followed by washing with 2× SSC for 5 minutes. The RP11-265B8 probe was detected with Avidin D-Texas Red, biotinylated anti-Avidin D and Avidin D-Texas Red (Vector Laboratories). The RP11-876N18 was detected with mouse anti-digoxigenin antibody (Sigma-Aldrich) followed by rabbit anti- mouse-FITC and anti- rabbit-FITC (Sigma-Aldrich). The slides were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) containing DAPI counterstain.
3
Multicolor Flow Cytometry Analysis of Stem Cell Markers
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10 For CD105, CD166 and CD34, primary mouse monoclonal antibodies and secondary antibodies rabbit anti-mouse FITC (Sigma, Milan, Italy) were used. For MHC-II, primary rat monoclonal antibody and secondary rabbit anti-rat FITC (Sigma) were used. Staining was performed as previously reported [40] . Cells (1x10 6 cells/mL) were labeled with primary antibodies in PBS with 3% of bovine serum albumin (BSA) (BDH; VWR International Ltd, Poole, UK) for 45 minutes at room temperature in the dark, followed by washing in cold PBS and a final incubation with secondary antibodies (1:50) for 30 minutes at room temperature in the dark. After incubation, cells were washed twice in ice-cold PBS and analyzed using a Millipore Guava easyCyte Single Sample Flow Cytometer. A minimum of 10,000 cells was acquired for each sample and analyzed in the FL1 channel.
The negative pattern was examined by applying the same cell suspension with the first incubation, and the result was included on the global compensation, in order to exclude auto fluorescence. A 488 nm filter was used in each analysis.
Off-line analyses of the flow cytometry standard (FCS) files were performed using Weasel software v.2.5 (http://en.bio-soft.net/other/WEASEL.htmL).
The negative pattern was examined by applying the same cell suspension with the first incubation, and the result was included on the global compensation, in order to exclude auto fluorescence. A 488 nm filter was used in each analysis.
Off-line analyses of the flow cytometry standard (FCS) files were performed using Weasel software v.2.5 (http://en.bio-soft.net/other/WEASEL.htmL).
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