Alexa Fluor® 647 anti-mouse CD326 (Ep-CAM) Antibody

Product Details

Verified Reactivity

Mouse

Antibody Type

Monoclonal

Host Species

Rat

Immunogen

TE-71 thymic epithelial cell line

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® 647 under optimal conditions.

Concentration

0.5 mg/ml

Storage & Handling

The CD326 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
IHC-F, 3D IHC - Verified

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 ≤ 0.25 µg per 10⁶ cells in 100 µl volume. For immunohistochemistry on frozen tissue sections, a concentration range of 2.5 - 5.0 µg/ml is suggested. For 3D immunohistochemistry on formalin-fixed tissues, a concentration of 5.0 µg/mL is suggested. It is recommended that the reagent be titrated for optimal performance for each application.

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

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

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Excitation Laser

Red Laser (633 nm)

Application Notes

Additional reported applications for clone G8.8 (for the relevant formats) include: immunohistochemistry of frozen sections: acetone fixed¹, with or without OCT embedding^2,4, and spatial biology (IBEX)^13,14.

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)

1. Farr A, et al. 1991. J. Histochem. Cytochem. 39:645. (FC, IHC)
2. Dooley J, et al. 2005. J. Immunol. 175:4331. (FC, IHC)
3. Hinterberger M, et. al. 2010. Nat. Immunol. 11:512. (FC) PubMed
4. Gracz AD, et al. 2010. Am J. Physiol Gastrointest Liver Physiol. 298:590. (IHC) PubMed
5. Nudel I, et al. 2011. J. Immunol. 186:891. PubMed
6. Morimoto H, et al. 2012. Biol Reprod. 86:148. PubMed
7. Ishii K, et al. 2012. Development. 139:1734. PubMed
8. Takehashi M, et al. 2012. Biol Reprod. 86:178. PubMed
9. Murakami R, et al. 2013. PLoS One. 8:73270. PubMed
10. Taguchi K, et al. 2014. Mol Cell Biol. 34:900. PubMed
11. Hirokawa Y, et al. 2014. Am J Physiol Gastrointerest Liver Physiol. 306:547. PubMed
12. Ding X, et al. 2015. Cancer Res. 75:330. PubMed
13. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
14. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

1. Gabitova-Cornell L, et al. 2020. Cancer Cell. 38(4):567-583.e11. PubMed
2. Sandovici I, et al. 2022. Dev Cell. 57:63. PubMed
3. Grégoire C, et al. 2022. Immunity. 55:1216. PubMed
4. Moskwa N, et al. 2022. Development. 149:. PubMed
5. Mueller JPJ, et al. 2023. Front Immunol. 14:1034032. PubMed
6. Bilate AM, et al. 2020. Immunity. 53(5):1001-1014.e20. PubMed
7. Davis F, et al. 2015. Proc Natl Acad Sci U S A. 112:5827. PubMed
8. Molaro A, et al. 2014. Genes Dev. 28:1544. PubMed
9. Biffi G, et al. 2018. Cancer Discov. 2:282. PubMed
10. Strunz M, et al. 2020. Nat Commun. 2.929861111. PubMed
11. McClendon J, et al. 2017. Am J Pathol. 10.1016/j.ajpath.2017.04.012. PubMed
12. Blijswijk J, et al. 2015. J Immunol. 194:307. PubMed
13. Paiva RA, et al. 2021. Cell Reports. 35(2):108967. PubMed
14. Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed
15. Riemondy KA, et al. 2019. JCI Insight. 5:. PubMed
16. Joshi PA, et al. 2019. Nat Commun. 10:1760. PubMed
17. Burns KA, et al. 2018. Endocrinology. 159:103. PubMed
18. Cheng Q, et al. 2020. Nat Nanotechnol. 0.842361111. PubMed
19. Progatzky F, et al. 2021. Nature. 599:125. PubMed
20. Cohen SB et al. 2018. Cell host & microbe. 24(3):439-446 . PubMed
21. Campbell C et al. 2018. Immunity. 48(6):1245-1257 . PubMed
22. Xiong H, et al. 2021. Theranostics. 11:1594. PubMed
23. Zhang J, et al. 2018. Sci Rep. 8:2373. PubMed
24. Dravis C et al. 2018. Cancer cell. 34(3):466-482 . PubMed
25. Madenspacher JH, et al. 2020. JCI Insight. 5:00. PubMed
26. Heijden M, et al. 2016. Nat Commun. 7:10916. PubMed
27. Moon S, et al. 2021. J Exp Med. 218:. PubMed
28. Hegab A, et al. 2015. Stem Cell Res. 15: 109-121. PubMed
29. Shaffer M, et al. 2010. PLoS One. 5:e12404. PubMed
30. Kolodkin-Gal D, et al. 2021. Gut. Online ahead of print. PubMed
31. Ma Z, et al. 2022. Methods Mol Biol. 2471:49. PubMed
32. Tang R, et al. 2020. Elife. 9:00. PubMed
33. Giraddi R, et al. 2015. Nat Commun. 6: 8487. PubMed
34. Bernstock JD, et al. 2019. J Allergy Clin Immunol. 145:358. PubMed
35. Wasik K, et al. 2015. Genes Dev. 29: 1403 - 1415. PubMed
36. Stewart AS, et al. 2021. Am J Physiol Gastrointest Liver Physiol. 321:G588. PubMed
37. Steele NG, et al. 2021. Clin Cancer Res. 27:2023. PubMed
38. Miragaia RJ, et al. 2018. Sci Rep. 8:685. PubMed
39. Satoh–Takayama N, et al. 2020. Immunity. 52(4):635-649. PubMed
40. Fang Y, et al. 2022. Development. 149:. PubMed
41. Liu Y, et al. 2020. Cell. 183(6):1665-1681.e18. PubMed
42. Gaiser M, et al. 2012. Proc Natl Acad Sci U S A. 1.034027778. PubMed
43. Kurashima Y, et al. 2021. Nat Commun. 12:1067. PubMed
44. Bretz N, et al. 2014. Immunol Lett. 161:140. PubMed
45. Giraddi RR et al. 2018. Cell reports. 24(6):1653-1666 . PubMed
46. Koyama M et al. 2019. Immunity. 51(5):885-898 . PubMed
47. Oni TE, et al. 2020. J Exp Med. :217. PubMed
48. Chung CY et al. 2019. Cell Rep. 29(2):495-510 . PubMed

RRID

AB_1134104 (BioLegend Cat. No. 118211)
AB_1134104 (BioLegend Cat. No. 118212)

Antigen Details

Structure

40 kD single-pass type 1 glycoprotein. 293 amino acids, with a 21 aa signal peptide, a 246 aa extracellular domain, a 21 aa transmembrane domain, and a 26 aa cytoplasmic domain. The extracellular domain contains two epidermal growth factor-like repeats.

Distribution

Expressed on majority of epithelial cell membranes with the exception of adult squamous cells of the skin and a few specific epithelial cell types.

Function

Mediates calcium-independent homophilic cell-cell adhesion.

Interaction

CD326 displays hemophilic binding.

Ligand/Receptor

CD305 (LAIR-1), CD306 (LAIR-2), and Ep-CAM.

Cell Type

Embryonic Stem Cells, Epithelial cells

Biology Area

Immunology, Stem Cells

Molecular Family

Adhesion Molecules, CD Molecules

Antigen References

1. Borkowski TA, et al. 1996. Eur. J. Immunol. 26:110.
2. Bergsagel PL, et al. 1992. J. Immunol. 148:590.

Gene ID

17075 View all products for this Gene ID

UniProt

View information about CD326 on UniProt.org

Documentation

• Technical data sheet
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol
• Ce3D™ Tissue Clearing Kit

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