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

Pricing & Availability
Clone
G8.8 (See other available formats)
Regulatory Status
RUO
Other Names
CD326, EGP40, MIC18, TROP1, KSA
Isotype
Rat IgG2a, κ
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Product Citations
publications
1_G8dot8_A647_081908.jpg
TE-71 (mouse thymic epithelial stromal cell line) stained with G8.8 Alexa Fluor® 647
  • 1_G8dot8_A647_081908.jpg
    TE-71 (mouse thymic epithelial stromal cell line) stained with G8.8 Alexa Fluor® 647
  • 2_G8dot8_A647_CD326_Antibody_2_121018
    Dissected C57/B6 mouse small intestine was immersed in 4% paraformaldehyde (PFA) overnight followed by 30% sucrose immersion overnight and frozen in OCT. Frozen section was blocked with 5% FBS and 5% mouse serum for 30 minutes at room temperature. Then the tissue section was stained with 2.5 µg/mL of anti-mouse Tubulin Beta 3 (clone AA10) Alexa Fluor® 488 (green) and 2.5 µg/mL of anti-mouse CD326 (clone G8.8) Alexa Fluor® 647 (red) overnight at 4°C. Nuclei were counterstained with DAPI (blue). The image was captured by 10X objective.
  • 3_G8dot8_A647_CD326_Ep-CAM_Antibody_3D_IHC_05112021.png
    Paraformaldehyde-fixed (4%), 500 μm-thick mouse thymus tissue section was processed according to the Ce3DTM Tissue Clearing Kit protocol (cat. no. 427701). The section was costained with anti-mouse CD3 Antibody (clone 17A2) Alexa Fluor® 594 at 5 µg/mL (green), and anti-mouse CD326 (Ep-CAM) Antibody (clone G8.8) Alexa Fluor® 647 at 5 µg/mL (magenta). The section was then optically cleared and mounted in a sample chamber. The image was captured with a 10X objective using Zeiss 780 confocal microscope and processed by Imaris image analysis software.
    Watch the video.
  • 4_45_Mouse_Lung_EpCAM_MHCII_CD169
    Confocal image of C57BL/6 mouse lung sample acquired using the IBEX method of highly multiplexed antibody-based imaging: EpCAM (blue) in Cycle 2, MHCII (magenta) in Cycle 2, and CD169 (green) in Cycle 4. Tissues were prepared using ~1% (vol/vol) formaldehyde and a detergent. Following fixation, samples are immersed in 30% (wt/vol) sucrose for cryoprotection. Images are courtesy of Drs. Andrea J. Radtke and Ronald N. Germain of the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
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118211 25 µg $124
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118212 100 µg $265
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Description

EpCAM (CD326) mediates calcium-independent homophilic cell to cell adhesion. It may also function as a growth factor receptor. It is thought to be involved in maintaining cells in position during proliferation. Expression of EpCAM seems to correlate inversely with the level of E-cadherin (CD324). EpCAM is considered important in tumor biology.

Product Details
Technical data sheet

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 embedding2,4, 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. Bilate AM, et al. 2020. Immunity. 53(5):1001-1014.e20. PubMed
  4. Davis F, et al. 2015. Proc Natl Acad Sci U S A. 112:5827. PubMed
  5. Molaro A, et al. 2014. Genes Dev. 28:1544. PubMed
  6. Biffi G, et al. 2018. Cancer Discov. 2:282. PubMed
  7. Strunz M, et al. 2020. Nat Commun. 2.929861111. PubMed
  8. McClendon J, et al. 2017. Am J Pathol. 10.1016/j.ajpath.2017.04.012. PubMed
  9. Blijswijk J, et al. 2015. J Immunol. 194:307. PubMed
  10. Paiva RA, et al. 2021. Cell Reports. 35(2):108967. PubMed
  11. Lin JR et al. 2018. eLife. 7 pii: e31657. PubMed
  12. Riemondy KA, et al. 2019. JCI Insight. 5:. PubMed
  13. Joshi PA, et al. 2019. Nat Commun. 10:1760. PubMed
  14. Burns KA, et al. 2018. Endocrinology. 159:103. PubMed
  15. Cheng Q, et al. 2020. Nat Nanotechnol. 0.842361111. PubMed
  16. Progatzky F, et al. 2021. Nature. 599:125. PubMed
  17. Cohen SB et al. 2018. Cell host & microbe. 24(3):439-446 . PubMed
  18. Campbell C et al. 2018. Immunity. 48(6):1245-1257 . PubMed
  19. Xiong H, et al. 2021. Theranostics. 11:1594. PubMed
  20. Zhang J, et al. 2018. Sci Rep. 8:2373. PubMed
  21. Dravis C et al. 2018. Cancer cell. 34(3):466-482 . PubMed
  22. Madenspacher JH, et al. 2020. JCI Insight. 5:00. PubMed
  23. Heijden M, et al. 2016. Nat Commun. 7:10916. PubMed
  24. Moon S, et al. 2021. J Exp Med. 218:. PubMed
  25. Hegab A, et al. 2015. Stem Cell Res. 15: 109-121. PubMed
  26. Shaffer M, et al. 2010. PLoS One. 5:e12404. PubMed
  27. Kolodkin-Gal D, et al. 2021. Gut. Online ahead of print. PubMed
  28. Ma Z, et al. 2022. Methods Mol Biol. 2471:49. PubMed
  29. Tang R, et al. 2020. Elife. 9:00. PubMed
  30. Giraddi R, et al. 2015. Nat Commun. 6: 8487. PubMed
  31. Bernstock JD, et al. 2019. J Allergy Clin Immunol. 145:358. PubMed
  32. Wasik K, et al. 2015. Genes Dev. 29: 1403 - 1415. PubMed
  33. Stewart AS, et al. 2021. Am J Physiol Gastrointest Liver Physiol. 321:G588. PubMed
  34. Steele NG, et al. 2021. Clin Cancer Res. 27:2023. PubMed
  35. Miragaia RJ, et al. 2018. Sci Rep. 8:685. PubMed
  36. Satoh–Takayama N, et al. 2020. Immunity. 52(4):635-649. PubMed
  37. Fang Y, et al. 2022. Development. 149:. PubMed
  38. Liu Y, et al. 2020. Cell. 183(6):1665-1681.e18. PubMed
  39. Gaiser M, et al. 2012. Proc Natl Acad Sci U S A. 1.034027778. PubMed
  40. Kurashima Y, et al. 2021. Nat Commun. 12:1067. PubMed
  41. Bretz N, et al. 2014. Immunol Lett. 161:140. PubMed
  42. Giraddi RR et al. 2018. Cell reports. 24(6):1653-1666 . PubMed
  43. Koyama M et al. 2019. Immunity. 51(5):885-898 . PubMed
  44. Oni TE, et al. 2020. J Exp Med. :217. PubMed
  45. Chung CY et al. 2019. Cell Rep. 29(2):495-510 . PubMed
RRID
AB_1134104 (BioLegend Cat. No. 118211)
AB_1134101 (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

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.

Go To Top Version: 5    Revision Date: 04/21/2022

For Research Use Only. Not for diagnostic or therapeutic use.

 

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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