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Alexa Fluor® 700 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
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Product Citations
publications
1_M5slash114_Alx700_020608
C57BL/6 mouse splenocytes were stained with anti-mouse I-A/I-E (clone M5/114.15.2)Alexa Fluor® 700 (filled histogram) or rat IgG2b, κ Alexa Fluor® 700 isotype control (open histogram).
  • 1_M5slash114_Alx700_020608
    C57BL/6 mouse splenocytes were stained with anti-mouse I-A/I-E (clone M5/114.15.2)Alexa Fluor® 700 (filled histogram) or rat IgG2b, κ Alexa Fluor® 700 isotype control (open histogram).
  • 34_Mouse_Spleen_F480_MHCII
    Confocal image of C57BL/6 mouse spleen sample acquired using the IBEX method of highly multiplexed antibody-based imaging: MHCII (blue) in Cycle 2 and F4/80 (magenta) in Cycle 2. 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).
  • 39_Mouse_Thymus_CD68_MHCII
    Confocal image of C57BL/6 mouse thymus sample acquired using the IBEX method of highly multiplexed antibody-based imaging: CD68 (cyan) in Cycle 3 and MHCII (red) in Cycle 3. 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).
  • 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).
  • 51_Mouse_Gut_EpCAM_CD31_MHCII
    Confocal image of C57BL/6 mouse small intestine sample acquired using the IBEX method of highly multiplexed antibody-based imaging: EpCAM (blue) in Cycle 1, CD31 (magenta) in Cycle 2, and MHCII (green) in Cycle 3. 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).
  • 55_Mouse_Liver_Ecadherin_MHCII
    Confocal image of C57BL/6 mouse liver sample acquired using the IBEX method of highly multiplexed antibody-based imaging: E-Cadherin (blue) in Cycle 1 and MHCII (magenta) 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).
See Alexa Fluor® 700 spectral data
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107621 25 µg 76€
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107622 100 µg 172€
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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

Reactivity
Mouse
Antibody Type
Monoclonal
Host Species
Rat
Immunogen
Activated C57BL/6 mouse spleen 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 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 million 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 633 nm / 635 nm. 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.

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Excitation Laser
Red Laser (633 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
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RRID
AB_493726 (BioLegend Cat. No. 107621)
AB_493727 (BioLegend Cat. No. 107622)

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

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.

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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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