Alexa Fluor® 700 anti-mouse/human CD44 Antibody

Product Details

Verified Reactivity

Mouse, Human

Reported Reactivity

Chimpanzee, Baboon, Cynomolgus, Rhesus, Squirrel Monkey, Horse, Cow, Pig, Dog, Cat

Antibody Type

Monoclonal

Host Species

Rat

Immunogen

Dexamethasone-induced myeloid leukemia M1 cells

Formulation

Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.

Preparation

The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 700 under optimal conditions.

Concentration

0.5 mg/ml

Storage & Handling

The CD44 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. The suggested use of this reagent is ≤ 0.25 µg per 10⁶ cells in 100 µl volume. It is highly recommended that the reagent be titrated for optimal performance for each application.

* Alexa Fluor® 700 has a maximum emission of 719 nm when it is excited at 633nm / 635nm. Prior to using Alexa Fluor® 700 conjugate for flow cytometric analysis, please verify your flow cytometer's capability of exciting and detecting the fluorochrome.

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

View full statement regarding label licenses

Excitation Laser

Red Laser (633 nm)

Application Notes

Clone IM7 has been reported to recognize an epitope common to alloantigens and all isoforms of CD44^17,18 that is located between amino acids 145 and 186²⁰. This clone has been verified for immunocytochemistry (ICC) and frozen immunohistochemistry (IHC-F). Additional reported applications (for the relevant formats) include: immunohistochemistry of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections^6,7, complement-mediated cytotoxicity¹, immunoprecipitation^1,3, in vivo inhibition of DTH^4,5, and spatial biology (IBEX)^23,24. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. No. 103046, 103065 - 103069).

Cross-reactivity to ferret has been reported by a collaborator, but not verified in house.

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

(PubMed link indicates BioLegend citation)

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RRID

AB_493712 (BioLegend Cat. No. 103025)
AB_493712 (BioLegend Cat. No. 103026)

Antigen Details

Structure

Variable splicing of CD44 gene generates many CD44 isoforms, 80-95 kD

Distribution

All leukocytes, epithelial cells, endothelial cells, hepatocytes, mesenchymal cells

Function

Leukocyte attachment and rolling on endothelial cells, stromal cells and ECM

Ligand/Receptor

Hyaluronan, MIP-1β, fibronectin, collagen

Cell Type

B cells, Endothelial cells, Epithelial cells, Leukocytes, Mesenchymal cells, Mesenchymal Stem Cells, Tregs

Biology Area

Cell Adhesion, Cell Biology, Immunology, Stem Cells

Molecular Family

Adhesion Molecules, CD Molecules

Antigen References

1. Barclay AN, et al. 1997. The Leukocyte Antigen FactsBook Academic Press.
2. Haynes BF, et al. 1991. Cancer Cells 3:347.
3. Goldstein LA, et al. 1989. Cell 56:1063.
4. Mikecz K, et al. 1995. Nat. Med. 1:558.
5. Hegde V, et al. 2008. J. Leukocyte Biol. 84:134.
6. Liu T, et al. 2009. Biol. Direct 4:40.

Gene ID

12505 View all products for this Gene ID 960 View all products for this Gene ID

UniProt

View information about CD44 on UniProt.org

Documentation

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