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Alexa Fluor® 594 anti-mouse CD4 Antibody

Pricing & Availability
Clone
GK1.5 (See other available formats)
Regulatory Status
RUO
Other Names
L3T4, T4
Isotype
Rat IgG2b, κ
Ave. Rating
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Product Citations
publications
A_GK1.5_A594_CD4_IHCF_040318
C57BL/6 mouse frozen lymph node section was fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature and blocked with 5% FBS plus 5% rat serum for 1 hour at room temperature. Then the section was stained with 2.5 µg/ml of CD4 (clone GK1.5) Alexa Fluor® 594 (red), 2.5 µg/ml of CD8 (clone 53-6.7) Alexa Fluor® 647 (green), and 2.5 µg/ml of B220 (clone RA3-6B2) Alexa Fluor® 488 (blue) overnight at 4°C. The image was captured by 10X objective.
  • A_GK1.5_A594_CD4_IHCF_040318
    C57BL/6 mouse frozen lymph node section was fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature and blocked with 5% FBS plus 5% rat serum for 1 hour at room temperature. Then the section was stained with 2.5 µg/ml of CD4 (clone GK1.5) Alexa Fluor® 594 (red), 2.5 µg/ml of CD8 (clone 53-6.7) Alexa Fluor® 647 (green), and 2.5 µg/ml of B220 (clone RA3-6B2) Alexa Fluor® 488 (blue) overnight at 4°C. The image was captured by 10X objective.
  • B_GK1-5_AF594_CD4_Antibody_FC_072414.jpg
    C57BL/6 mouse splenocytes were stained with CD4 (clone GK1.5) Alexa Fluor® 594 (filled histogram). The data was acquired by BD LSRFortessa™ cell analyzer equipped with Yellow-Green Laser (561 nm).
  • C_35_Mouse_Spleen_CD4_CD8_IgD
    Confocal image of C57BL/6 mouse spleen sample acquired using the IBEX method of highly multiplexed antibody-based imaging: CD4 (cyan), CD8 (magenta), and IgD (blue) in Cycle 1. 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 Alexa Fluor® 594 spectral data
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100446 100 µg $253
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Description

CD4 is a 55 kD protein also known as L3T4 or T4. It is a member of the Ig superfamily, primarily expressed on most thymocytes, a subset of T cells, and weakly on macrophages and dendritic cells. It acts as a coreceptor with the TCR during T cell activation and thymic differentiation by binding MHC class II and associating with the protein tyrosin kinase, lck.

Product Details
Technical data sheet

Product Details

Verified Reactivity
Mouse
Antibody Type
Monoclonal
Host Species
Rat
Immunogen
Mouse CTL clone V4
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® 594 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

IHC-F - Quality tested
FC - Verified

SB - Reported in the literature, not verified in house
Recommended Usage

Each lot of this antibody is quality control tested by immunohistochemistry. For immunohistochemistry on frozen tissue sections, a concentration range of 2.5-5 μg/ml is suggested. For flow cytometric staining, 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.

* Alexa Fluor® 594 has an excitation maximum of 590 nm, and a maximum emission of 617 nm.


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

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Application Notes

Additional reported applications (for the relevant formats) include: blocking of CD4+ T cell activation1,4,11, thymocyte costimulation3, in vitro and in vivo depletion2,5-8, blocking of egg-sperm cell adhesion1,4, immunohistochemical staining of acetone-fixed frozen sections9,10, immunoprecipitation1,2, and spatial biology (IBEX)12,13. The GK1.5 antibody is able to block CD4 mediated cell adhesion and T cell activation. Binding of GK1.5 antibody to CD4 T cells can be blocked by RM4-5 antibody, but not RM4-4 antibody. For in vivo studies or highly sensitive assays, we recommend Ultra-LEAF™ purified antibody (Cat. No. 100442) with a lower endotoxin limit than standard LEAF™ purified antibodies (Endotoxin < 0.01 EU/µg).

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. Dialynas DP, et al. 1983. J. Immunol. 131:2445. (Block, IP)
  2. Dialynas DP, et al. 1983. Immunol. Rev. 74:29. (IP, Deplete)
  3. Wu L, et al. 1991. J. Exp. Med. 174:1617. (Costim)
  4. Godfrey DI, et al. 1994. J. Immunol. 152:4783. (Block)
  5. Gavett SH, et al. 1994. Am. J. Respir. Cell. Mol. Biol. 10:587. (Deplete)
  6. Schuyler M, et al. 1994. Am. J. Respir. Crit. Care Med. 149:1286. (Deplete)
  7. Ghobrial RR, et al. 1989. Clin. Immunol. Immunopathol. 52:486. (Deplete)
  8. Israelski DM, et al. 1989. J. Immunol. 142:954. (Deplete)
  9. Zheng B, et al. 1996. J. Exp. Med. 184:1083. (IHC)
  10. Frei K, et al. 1997. J. Exp. Med. 185:2177. (IHC)
  11. Felix NJ, et al. 2007. Nat. Immunol. 8:388. (Block)
  12. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
  13. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations
  1. Feist M, et al. 2021. Cancer Gene Ther. 28:98. PubMed
  2. Wang D, et al. 2018. Immunity. 48:659. PubMed
  3. Baptista AP et al. 2019. Immunity. 50(5):1188-1201 . PubMed
  4. Wang B, et al. 2022. Nat Commun. 13:3821. PubMed
  5. Ji Y, et al. 2017. Mucosal Immunol. 10.1038/mi.2016.119. PubMed
  6. Bosnjak B, et al. 2019. Front Immunol. 10:840. PubMed
  7. Kovacs SB, et al. 2021. STAR Protoc. 2:100244. PubMed
  8. Tanaka Y, et al. 2017. J Immunol. 199:4016. PubMed
  9. Kovacs SB, et al. 2020. Cell Reports. 32(4):107967. PubMed
  10. Liu Q, et al. 2021. Adv Mater. 33:e2102852. PubMed
  11. Wang X, et al. 2021. Sci Transl Med. 13:. PubMed
  12. Chmielewski M and Abken H 2017. Cell Rep.. 10.1016/j.celrep.2017.11.063. PubMed
RRID
AB_2563182 (BioLegend Cat. No. 100446)

Antigen Details

Structure
Ig superfamily, 55 kD
Distribution

Majority of thymocytes, T cell subset

Function
TCR co-receptor, T cell activation
Ligand/Receptor
MHC class II molecule
Cell Type
Dendritic cells, T cells, Thymocytes, Tregs
Biology Area
Immunology
Molecular Family
CD Molecules
Antigen References

1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press.
2. Bierer BE, et al. 1989. Annu. Rev. Immunol. 7:579.
3. Janeway CA. 1992. Annu. Rev. Immunol. 10:645.

Gene ID
12504 View all products for this Gene ID
UniProt
View information about CD4 on UniProt.org

Related FAQs

I am unable to see expression of T cell markers such as CD3 and CD4 post activation.
TCR-CD3 complexes on the T-lymphocyte surface are rapidly downregulated upon activation with peptide-MHC complex, superantigen or cross-linking with anti-TCR or anti-CD3 antibodies. PMA/Ionomycin treatment has been shown to downregulate surface CD4 expression. Receptor downregulation is a common biological phenomenon and so make sure that your stimulation treatment is not causing it in your sample type.
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 CD4 Reagents Request Custom Conjugation
Description Clone Applications
APC anti-mouse CD4 GK1.5 FC
Biotin anti-mouse CD4 GK1.5 FC,IHC-F,ICC
FITC anti-mouse CD4 GK1.5 FC,IHC-F,ICC
PE anti-mouse CD4 GK1.5 FC
PE/Cyanine5 anti-mouse CD4 GK1.5 FC
Purified anti-mouse CD4 GK1.5 FC,IHC-F,ICC,IP,Costim,Block,Depletion
PE/Cyanine7 anti-mouse CD4 GK1.5 FC
APC/Cyanine7 anti-mouse CD4 GK1.5 FC
Alexa Fluor® 647 anti-mouse CD4 GK1.5 FC,IHC-F,ICC
Alexa Fluor® 488 anti-mouse CD4 GK1.5 FC,IHC-F,ICC
Pacific Blue™ anti-mouse CD4 GK1.5 FC
Alexa Fluor® 700 anti-mouse CD4 GK1.5 FC
PerCP anti-mouse CD4 GK1.5 FC
PerCP/Cyanine5.5 anti-mouse CD4 GK1.5 FC
Brilliant Violet 421™ anti-mouse CD4 GK1.5 FC,ICC,IHC-F
Ultra-LEAF™ Purified anti-mouse CD4 GK1.5 FC,Block,Costim,Depletion,IHC,IP
Alexa Fluor® 594 anti-mouse CD4 GK1.5 IHC-F,FC,ICC,SB
Brilliant Violet 711™ anti-mouse CD4 GK1.5 FC
Brilliant Violet 510™ anti-mouse CD4 GK1.5 FC,IHC-F,ICC
Brilliant Violet 605™ anti-mouse CD4 GK1.5 FC
Brilliant Violet 785™ anti-mouse CD4 GK1.5 FC
PE/Dazzle™ 594 anti-mouse CD4 GK1.5 FC
APC/Fire™ 750 anti-mouse CD4 GK1.5 FC
GoInVivo™ Purified anti-mouse CD4 GK1.5 FC
Brilliant Violet 750™ anti-mouse CD4 GK1.5 FC
Brilliant Violet 650™ anti-mouse CD4 GK1.5 FC
Spark Blue™ 550 anti-mouse CD4 GK1.5 FC
Spark NIR™ 685 anti-mouse CD4 GK1.5 FC
KIRAVIA Blue 520™ anti-mouse CD4 GK1.5 FC
PE/Fire™ 640 anti-mouse CD4 GK1.5 FC
APC/Fire™ 810 anti-mouse CD4 GK1.5 FC
PE/Fire™ 700 anti-mouse CD4 GK1.5 FC
Spark Violet™ 538 anti-mouse CD4 GK1.5 FC
Spark YG™ 593 anti-mouse CD4 GK1.5 FC
Spark Blue™ 574 anti-mouse CD4 Antibody GK1.5 FC
Spark UV™ 387 anti-mouse CD4 GK1.5 FC
Spark Blue™ 515 anti-mouse CD4 GK1.5 FC
Spark PLUS UV395™ anti-mouse CD4 GK1.5 FC
Spark Red™ 718 anti-mouse CD4 (Flexi-Fluor™) GK1.5 FC
Go To Top Version: 3    Revision Date: 04/21/2022

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

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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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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