C57BL/6 mouse splenocytes stained with N418 Alexa Fluor® 647
C57BL/6 mouse splenocytes stained with N418 Alexa Fluor® 647
Paraformaldehyde-fixed (4%), 500 μm-thick mouse spleen section was processed according to the Ce3DTM Tissue Clearing Kit protocol (cat. no. 427701). The section was costained with anti-mouse/human CD45R/B220 Antibody (clone RA3-6B2) Alexa Fluor® 488 at 5 µg/mL (green), anti-mouse CD8a Antibody (clone 53-6.7) Alexa Fluor® 594 at 5 µg/mL (yellow), and anti-mouse CD11c Antibody (clone N418) 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.
Fixed whole mount mouse epidermis Langerhans cells were stained with Alexa Fluor® 647 CD11c (red) (clone N418) and PE CD207 (yellow) (clone 4C7). Isotype controls at the same concentrations were used for the negative control. Cells were mounted in Prolong Gold and imaged with a Leica SP8 confocal. Image courtesy of Grzegorz Chodaczek and Zbigniew Mikulski at LIAI.
Live intravital mouse spleen imaging. PE CD11b (red) (clone M1/70), Alexa Fluor® 488 F4/80 (green) (clone BM8), and Alexa Fluor® 647 CD11c (blue) (clone N418) were imaged 30 minutes after iv injection of 10 µg per antibody. Isotype controls at the same concentrations, time post injection, and exposure conditions were used for the negative control. Cells were imaged with a Leica SP5 confocal on anesthetized mice. Image courtesy of Grzegorz Chodaczek and Zbigniew Mikulski at LIAI.
Fixed whole mount mouse spleen was stained with FITC CD172a (red) (clone P84), Alexa Fluor® CD11c (yellow) (clone N418), and PE CD11b (blue) (clone M1/70). Isotype controls at the same concentrations were used for the negative control. Cells were mounted in Prolong Gold and imaged with a Leica SP8 confocal. Image courtesy of Grzegorz Chodaczek and Zbigniew Mikulski at LIAI.
Confocal image of C57BL/6 mouse spleen sample acquired using the IBEX method of highly multiplexed antibody-based imaging: CD11c (cyan) in Cycle 2 and CD68 (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).
CD11c is a 150 kD glycoprotein also known as αX integrin, CR4, and p150. CD11c forms a αXβ2 heterodimer with β2 integrin (CD18). It is primarily expressed on dendritic cells, NK cells, a subset of intestinal intraepithelial lymphocytes (IEL), and some activated T cells. The αXβ2 integrin plays an important role in cell-cell contact by binding its ligands: iC3b, fibrinogen, and CD54.
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 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 other applications.
* 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.
Additional reported applications (for the relevant formats) include: immunoprecipitation3, immunohistochemical staining of acetone-fixed frozen sections3, immunofluorescence microscopy5, 9 (Alexa Fluor® 488 conjugated N418 was used for IHC in frozen sections10), and spatial biology (IBEX)22,23.
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
Granucci F, et al. 1997. J. Immunol. 159:1794.
Stokes RW, et al. 1998. J. Immunol. 160:5514.
Metlay JP, et al. 1990. J. Exp. Med. 171:1753. (IHC, IP)
Ma XT, et al. 2006. Cancer Research 66:1169.
Chin RK, et al. 2006. J. Immunol. 177:290. (IF)
Cervantes-Barragan L, et al. 2007. Blood 109:1131. (FC) PubMed
Turnquist HR, et al. 2007. J. Immunol. 178:7018. (FC) PubMed
Benson MJ, et al. 2007. J. Exp. Med. doi:10.1084/jem.20070719. (FC) PubMed
You Y, et al. 2009. J. Immunol. 182:7343. (IF) PubMed
Roland CL, et al. 2009. Mol. Cancer Res. 8:1761. (IHC, FC) PubMed
Wikstrom M, et al.2006. J. Immunol. 177:913. PubMed
Pericolini E, et al. 2008. J. Leukocyte Biol. 83:1286. PubMed
Randall LM, et al. 2008. Infect. Immun.76:3312. PubMed
Fahlen-Yrild L, et al. 2009. J. Immunol. 183:5032. PubMed
Osterholzer JJ, et al. 2009. J. Immunol. 183:8044. PubMed
Bankoti J, et al. 2010. Toxicol. Sci. 115:422. (FC) PubMed
Eisenach PA, et al. 2010. J Cell Sci. 123:4182. PubMed
1. Barclay A, et al. 1997. The Leukocyte Antigen Facts Book Academic Press.
2. Springer TA. 1994. Cell 76:301.
3. Lopez-Rodriguez C, et al. 1996. J. Immunol. 156:3780.
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.
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