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Immunogold and electron microscopic localization

KB Kevin Boyé
NP Nadège Pujol
IA Isabel D Alves
YC Ya-Ping Chen
TD Thomas Daubon
YL Yi-Zong Lee
SD Stephane Dedieu
MC Marion Constantin
LB Lorenzo Bello
MR Marco Rossi
RB Rolf Bjerkvig
SS Shih-Che Sue
AB Andreas Bikfalvi
CB Clotilde Billottet
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Subconfluent U87-CXCR3-A cells stimulated 60 min with CXCR3 agonist were fixed in a mixture of 2% paraformaldehyde and 0.1% glutaraldéhyde in 0.1 M sodium phosphate buffer, pH 7.4 for 1 h and kept in 0.1 M sodium phosphate buffer until further processing. Cell samples were embedded in 10% gelatin, which was solidified on ice. Blocks with cells were immersed in 2.3 M sucrose in phosphate buffer at 4 °C, and ultra-thin cryosections were single or double immunolabeled with 6- and 15-nm protein A-conjugated colloidal gold probes. The rabbit antibodies used were against syntaxin 6 (6 nm, ref: 2869 Cell Signaling, dilution: 1/10) and the mouse antibodies used were agiants CXCR3 (15 nm, ref: 2ar1 ab64714, dilution: 1/300). The labeled sections were contrasted with uranyl acetate and embedded in methyl cellulose.

Copyright and License information: The Author(s) ©2017
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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