Alexa Fluor® 647 anti-human CD54 Antibody

Product Details

Verified Reactivity

Human

Antibody Type

Monoclonal

Host Species

Mouse

Immunogen

Colonic cancer BM314 cells

Formulation

Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA)

Preparation

The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.

Concentration

Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

Storage & Handling

The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.

Application

FC - Quality tested
SB - Reported in the literature, not verified in house

Recommended Usage

Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood.

* Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm.

Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.

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Excitation Laser

Red Laser (633 nm)

Application Notes

Clone HA58 recognizes an epitope located in the extracellular D1 domain of CD54³. Additional reported applications (for the relevant formats) include: spatial biology (IBEX)^4,5.

Additional Product Notes

Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).

Application References

1. Tsujisaki M, et al. 1991. Clin. Exp. Immunol. 85:3.
2. Kanwar JR, et al. 2003. Cancer Gene Ther. 10:468.
3. Kohka H, et al. 1998. J. Leukoc. Biol. 64:519.
4. Radtke AJ, et al. 2020. Proc Natl Acad Sci USA. 117:33455-33465. (SB) PubMed
5. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

1. Zhang W, et al. 2022. Dev Cell. 57:329. PubMed

RRID

AB_2715941 (BioLegend Cat. No. 353113)
AB_2715941 (BioLegend Cat. No. 353114)

Antigen Details

Structure

Type I membrane protein, Ig superfamily, 85-110 kD

Distribution

Endothelial cells, T cells and B cells, monocytes/macrophages, granulocytes, and dendritic cells

Cell Type

B cells, Dendritic cells, Endothelial cells, Granulocytes, Macrophages, Mesenchymal Stem Cells, Monocytes, T cells

Biology Area

Cell Adhesion, Cell Biology, Costimulatory Molecules, Immunology, Neuroscience, Neuroscience Cell Markers, Stem Cells

Molecular Family

Adhesion Molecules, CD Molecules

Antigen References

1. Voraberger G, et al. 1991. J. Immunol. 147:2777.
2. Staunton DE, et al. 1988. Cell 52:925.
3. Greve JM, et al. 1989. Cell 56:839.

Gene ID

3383 View all products for this Gene ID

UniProt

View information about CD54 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.

Are other fluorophores compatible with IBEX?

Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.

The same antibody works in one tissue type but not another. What is happening?

Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.

How can I be sure the staining I’m seeing in my tissue is real?

In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.

Other Formats

Certificate of Analysis