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Brilliant Violet 421™ anti-mouse I-A/I-E Antibody

Pricing & Availability
Clone
M5/114.15.2 (See other available formats)
Regulatory Status
RUO
Other Names
MHC class II
Isotype
Rat IgG2b, κ
Ave. Rating
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Product Citations
publications
1_M5slash114_BV421_032911
C57BL/6 mouse splenocytes were stained with mouse I-A/I-E (clone M5/114.15.2) Brilliant Violet 421™ (filled histogram) or rat IgG2b, κ Brilliant Violet 421™ isotype control (open histogram).
  • 1_M5slash114_BV421_032911
    C57BL/6 mouse splenocytes were stained with mouse I-A/I-E (clone M5/114.15.2) Brilliant Violet 421™ (filled histogram) or rat IgG2b, κ Brilliant Violet 421™ isotype control (open histogram).
  • 2_M5slash114_BV421_071112
    BL/6 mouse lymph nodes, fixed O/N in PLP, blocked with 10% rat serum, stained with I-A/I-E BV421™ (red), B220 Alexa Fluor® 647 (blue), and CD11c FITC (green) in 1% BSA and 0.1% Tween-20 in PBS. Images were acquired with an automated widefield microscope (Nikon Eclipse Ti) and a CCD camera (QImaging Retiga 2000R). Emitted light was collected through 440/40, 525/50, and 700/75 nm bandpass filters. Images provided by Ann Haberman and Christine Podolski, Yale University.
  • 3_57_Mouse_Lymph_Node_CD3_MHCII
    Mice were injected subcutaneously with sheep red blood cells in a volume of 25 µl per site on days 0 and 4 and harvested on day 11. Confocal image of C57BL/6 mouse lymph node acquired using the IBEX method of highly multiplexed antibody-based imaging: CD3 (cyan) in Cycle 1 and MHCII (blue) in Cycle 10. 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).
Compare all formats See Brilliant Violet 421™ spectral data
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107631 125 µL 176 CHF
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107632 50 µg 237 CHF
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Description

These class II molecules are expressed on antigen presenting cells (including B cells) and a subset of T cells from H-2b,d,q,r bearing mice and are involved in antigen presentation to T cells expressing CD3/TCR and CD4 proteins.

Product Details
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Mouse
Antibody Type
Monoclonal
Host Species
Rat
Immunogen
Activated C57BL/6 mouse spleen cells
Formulation
µl size: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA).
µg size: 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 Brilliant Violet 421™ under optimal conditions.
Concentration
µg sizes: 0.2 mg/mL
µL sizes: 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
IHC-F - 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 using the µl size, 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. For flow cytometric staining using the µg size, the suggested use of this reagent is ≤ 0.25 µg per million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.

Brilliant Violet 421™ excites at 405 nm and emits at 421 nm. The standard bandpass filter 450/50 nm is recommended for detection. Brilliant Violet 421™ is a trademark of Sirigen Group Ltd.


Learn more about Brilliant Violet™.

This product is subject to proprietary rights of Sirigen Inc. and is made and sold under license from Sirigen Inc. The purchase of this product conveys to the buyer a non-transferable right to use the purchased product for research purposes only. This product may not be resold or incorporated in any manner into another product for resale. Any use for therapeutics or diagnostics is strictly prohibited. This product is covered by U.S. Patent(s), pending patent applications and foreign equivalents.
Excitation Laser
Violet Laser (405 nm)
Application Notes

The M5/114.15.2 antibody reacts with a polymorphic determinant shared by the I-Ab, I-Ad, I-Aq, I-Ed, and I-Ek MHC class II alloantigens from mice carrying H-2p,r,q,b,d,u haplotypes. Clone M5/114.15.2 however does not react wtih I-Af, I-Ak, or I-As MHC class II alloantigens.1

Additional reported applications (for the relevant formats) include: immunoprecipitation1, immunohistochemistry of frozen sections2,3,6, in vitro and in vivo blocking of antigen presentation or ligand binding4-7, and spatial biology (IBEX)17,18. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. Nos. 107655 & 107656).

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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  2. Viville S, et al. 1993. Cell 72:635. (IHC)
  3. Nelson AJ, et al. 1993. J. Immunol. 151:2453. (IHC)
  4. Shi Y, et al. 1998. J. Exp. Med. 187:367. (Block)
  5. Yamashita I, et al. 1993. Int. Immunol. 5:1139.
  6. Guo M, et al. 1995. Zygote 3:65. (IHC)
  7. Kim A, et al. 2004. Exp. Mol. Med. 36:428. (Block)
  8. Luckashenak NA, et al. 2006. J. Immunol. 177:5177.
  9. Venanzi ES, et al. 2007. J. Immunol. 179:5693.
  10. Christensen SR, et al. 2006. Immunity 25:417. PubMed
  11. Matte-Martone C, et al. 2008. Blood 111:3884. PubMed
  12. De Pascalis R, et al. 2008. Infect. Immun. 76:4311. PubMed
  13. Kuns RD, et al. 2009. Blood 113:5999. PubMed
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  16. Fu H, et al. 2014. Nat Commun. 5:3436. PubMed
  17. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
  18. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations
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  7. Roussel-Queval A, et al. 2023. STAR Protoc. 4:102119. PubMed
  8. Pasqual G, et al. 2023. Methods Mol Biol. 2618:71. PubMed
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  18. Ostendorf BN, et al. 2020. Nat Med. 26:1048. PubMed
  19. Perner C, et al. 2020. Immunity. 53(5):1063-1077.e7. PubMed
  20. Mariani SA, et al. 2019. Immunity. 50:1439. PubMed
  21. Voisin M, et al. 2021. Commun Biol. 4:420. PubMed
  22. Wang F, et al. 2021. Cell Mol Gastroenterol Hepatol. 13:257. PubMed
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  24. Mehta AK, et al. 2021. Nat Cancer. 2:66. PubMed
  25. Ferreira R, et al. 2016. J Infect Dis. 213: 669 - 673. PubMed
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  28. Roberts LM, et al. 2021. iScience. 24:103025. PubMed
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  43. Matsuda T, et al. 2022. iScience. 25:105324. PubMed
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  48. Lai SM et al. 2018. Cell reports. 25(11):3099-3109 . PubMed
  49. Zhang Q, et al. 2022. Commun Biol. 5:544. PubMed
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RRID
AB_10900075 (BioLegend Cat. No. 107631)
AB_10900075 (BioLegend Cat. No. 107632)

Antigen Details

Structure
MHC class II
Distribution

B cell and activated T cells, APCs of the H-2b,d,q,r bearing mice

Function
Antigen presentation
Ligand/Receptor
CD3/TCR, CD4
Cell Type
Antigen-presenting cells, B cells, Dendritic cells, T cells, Tregs
Biology Area
Immunology, Innate Immunity
Molecular Family
MHC Antigens
Antigen References

1. Watts C. 1997. Ann. Rev. Immunol. 15:821.
2. Pamer E, et al. 1998. Ann. Rev. Immunol. 16:323.

Gene ID
14961 View all products for this Gene ID 14969 View all products for this Gene ID
UniProt
View information about I-A/I-E on UniProt.org

Related FAQs

What is the F/P ratio range of our BV421™ format antibody reagents?

It is lot-specific. On average it ranges between 2-4.

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

View All I-A/I-E Reagents Request Custom Conjugation
Description Clone Applications
Biotin anti-mouse I-A/I-E M5/114.15.2 FC
FITC anti-mouse I-A/I-E M5/114.15.2 FC
PE anti-mouse I-A/I-E M5/114.15.2 FC
Purified anti-mouse I-A/I-E M5/114.15.2 FC,IHC-F,IP,Block
PE/Cyanine5 anti-mouse I-A/I-E M5/114.15.2 FC
APC anti-mouse I-A/I-E M5/114.15.2 FC
Alexa Fluor® 488 anti-mouse I-A/I-E M5/114.15.2 FC,3D IHC
Alexa Fluor® 647 anti-mouse I-A/I-E M5/114.15.2 FC
Pacific Blue™ anti-mouse I-A/I-E M5/114.15.2 FC
Alexa Fluor® 700 anti-mouse I-A/I-E M5/114.15.2 FC,SB
PerCP/Cyanine5.5 anti-mouse I-A/I-E M5/114.15.2 FC
PerCP anti-mouse I-A/I-E M5/114.15.2 FC
APC/Cyanine7 anti-mouse I-A/I-E M5/114.15.2 FC
PE/Cyanine7 anti-mouse I-A/I-E M5/114.15.2 FC
Brilliant Violet 421™ anti-mouse I-A/I-E M5/114.15.2 FC,IHC-F,SB
Brilliant Violet 510™ anti-mouse I-A/I-E M5/114.15.2 FC
Purified anti-mouse I-A/I-E (Maxpar® Ready) M5/114.15.2 FC,CyTOF®
Brilliant Violet 605™ anti-mouse I-A/I-E M5/114.15.2 FC
Brilliant Violet 650™ anti-mouse I-A/I-E M5/114.15.2 FC
Brilliant Violet 711™ anti-mouse I-A/I-E M5/114.15.2 FC
Brilliant Violet 785™ anti-mouse I-A/I-E M5/114.15.2 FC
PE/Dazzle™ 594 anti-mouse I-A/I-E M5/114.15.2 FC
Alexa Fluor® 594 anti-mouse I-A/I-E M5/114.15.2 IHC-F,3D IHC
APC/Fire™ 750 anti-mouse I-A/I-E M5/114.15.2 FC
TotalSeq™-A0117 anti-mouse I-A/I-E M5/114.15.2 PG
Ultra-LEAF™ Purified anti-mouse I-A/I-E M5/114.15.2 FC,IHC-F,IP,Block
TotalSeq™-B0117 anti-mouse I-A/I-E M5/114.15.2 PG
TotalSeq™-C0117 anti-mouse I-A/I-E M5/114.15.2 PG
Spark Blue™ 550 anti-mouse I-A/I-E M5/114.15.2 FC
PE/Fire™ 640 anti-mouse I-A/I-E M5/114.15.2 FC
Spark YG™ 581 anti-mouse I-A/I-E M5/114.15.2 FC
PE/Fire™ 810 anti-mouse I-A/I-E M5/114.15.2 FC
Spark UV™ 387 anti-mouse I-A/I-E M5/114.15.2 FC
Spark Violet™ 538 anti-mouse I-A/I-E M5/114.15.2 FC
PerCP/Fire™ 806 anti-mouse I-A/I-E M5/114.15.2 FC
Spark Red™ 718 anti-mouse I-A/I-E M5/114.15.2 FC
APC/Fire™ 810 anti-mouse I-A/I-E M5/114.15.2 FC
Spark PLUS UV395™ anti-mouse I-A/I-E M5/114.15.2 FC
Go To Top Version: 6    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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