Brilliant Violet 421™ anti-mouse CD68 Antibody

Product Details

Verified Reactivity

Mouse

Antibody Type

Monoclonal

Host Species

Rat

Immunogen

Purified Con A receptor glycoproteins from the P815 cell line

Formulation

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

0.2 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

ICFC - 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 intracellular immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤0.5 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

Additional reported (for relevant formats) applications include: immunoprecipitation^{1, 2}, Western Blot^{1, 2}, immunohistochemical staining of frozen sections² and paraformaldehyde-fixed paraffin-embedded sections³, and spatial biology (IBEX)^9,10.

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. Silva RP, et al. 1999. Biochem. J. 338:687. (IP, WB)
2. Rabinowitz SS, et al. 1991. J. Exp. Med. 174:827. (IP, WB, IHC)
3. Wu J, et al. 2008. P. Natl. Acad. Sci. USA 105:16934. (IHC)
4. Kayama H, et al. 2012. PNAS. 109:5010. PubMed
5. Park S, et al. 2013. Biomaterials. 34:598. PubMed
6. Guiducci C, et al. 2013. J Exp Med. 210:2903. PubMed
7. McKinstry SU, et al. 2014. J Neurosci. 34:9455. PubMed
8. Li X, et al. 2015. J Am Heart Assoc. 6:4. PubMed
9. Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
10. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

1. Chen J, et al. 2022. Adv Sci (Weinh). 9:e2200668. PubMed
2. Gomez-Salinero JM, et al. 2022. Cell Stem Cell. 29:593. PubMed
3. Bonifacio JPPL, et al. 2022. J Virol. 96:e0087122. PubMed
4. Cai B, et al. 2021. Mol Cancer. 20:165. PubMed
5. Socodato R, et al. 2020. Sci Signal. 13: . PubMed
6. Gangoso E, et al. 2021. Cell. 184:2454. PubMed

RRID

AB_2562949 (BioLegend Cat. No. 137017)

Antigen Details

Structure

A member of the lysosomal-associated membrane protein (lamp) family.

Distribution

Expressed on tissue macrophages, Langerhans cells, and at low levels on dendritic cells.

Function

Involved in cell-cell interaction or cell-ligand interaction, still not completely understood.

Cell Type

Antigen-presenting cells, Dendritic cells, Langerhans cells, Leukocytes, Macrophages

Biology Area

Cell Biology, Immunology, Innate Immunity, Neuroscience, Neuroscience Cell Markers

Molecular Family

Adhesion Molecules, CD Molecules, Innate Immune Signaling

Antigen References

1. Ramprasad MP, et al. 1996. Proc. Natl. Acad. Sci. USA 93:14833.
2. Smith MJ, et al. 1987. J. Cell. Sci. 87:113.

Gene ID

12514 View all products for this Gene ID

UniProt

View information about CD68 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Surface and Intracellular Cytokine Staining for Flow Cytometry - Video
• Intracellular Flow Cytometry Staining Protocol

Related Pages & Pathways

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

Certificate of Analysis