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Immunofluorescence staining of human U-2 OS cells. Cultured cells from the human U-2 OS (Osteosarcoma, ATCC HTB-96) cell line were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized (5 min) with BD Phosflow™ Perm Buffer III (Cat. No. 558050), washed with 1x PBS, and blocked (30 min) with 5% Goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1x PBS. The cells were stained (1 hr) with Purified Rabbit Anti-PCNA antibody (Abcam) at 2.5 μg/mL. After washing, the cells were counterstained (45 min) in blocking buffer with the second step reagent, BD Horizon™ BV480 Anti- Rabbit IgG (Cat. No. 564879; 2.5 μg/mL; pseudo-colored green). Cells were then stained (1 hr) in blocking buffer with Alexa Fluor® 488 Mouse Anti-β-Tubulin (Cat. No. 558605; pseudo-colored red). BD Pharmingen DAPI Solution (Cat. No. 564907) was used as a nuclear counterstain (pseudo-colored blue). Slides were mounted with ProLong® Gold and the image was captured on a BD Pathway™ 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision™ Software. 20X objective.


BD Horizon™ BV480 Goat Anti-Rabbit IgG

Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349).
For Immunofluorescence Applications:
The use of a mounting reagent (eg, ProLong® Gold) is highly recommended to maximize the photostability of BV480. For confocal microscopy systems, a 440 nm laser is the optimal excitation source and the recommended emission filter is a 485/20 nm bandpass filter.
For epifluorescence microscopes with broad spectrum excitation sources, the recommended excitation and emission filters are 445/20 nm and 485/20 nm bandpass filters, respectively. For specific multicolor imaging applications, the exact filter configurations should be optimized by the end user. For additional instrument/filter configuration information, please visit http://www.bdbiosciences.com/research/cellularimaging.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- BD Horizon Brilliant Violet 480 is covered by one or more of the following US patents: 8,575,303; 8,354,239.
- ProLong® is a registered trademark of Thermo Fisher Scientific, Inc. Waltham, MA.
- Triton is a trademark of the Dow Chemical Company.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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BD Horizon™ BV480 Goat Anti-Rabbit IgG is intended to be a second-step reagent for immunofluorescent staining of cells pre-stained with Rabbit IgG primary antibodies. It reacts with rabbit IgG and the light chains of other rabbit immunoglobulins. BV480 Anti-Rabbit IgG has minimal crossreactivity with human, mouse, or rat serum proteins. As a second step staining reagent, it is reactive with rabbit polyclonal and monoclonal antibodies.
The antibody was conjugated to BD Horizon BV480 which is part of the BD Horizon Brilliant™ Violet family of dyes. With an Ex Max of 436-nm and Em Max at 478-nm, BD Horizon BV480 can be excited by the violet laser and detected in the BD Horizon BV510 (525/40-nm) filter set. BV480 has less spillover into the BV605 detector and, in general, is brighter than BV510.

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.