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Histology and immunohistochemistry

JD Johanne Dubail
CH Céline Huber
SC Sandrine Chantepie
SS Stephan Sonntag
BT Beyhan Tüysüz
EM Ercan Mihci
CG Christopher T. Gordon
ES Elisabeth Steichen-Gersdorf
JA Jeanne Amiel
BN Banu Nur
IS Irene Stolte-Dijkstra
AE Albertien M. van Eerde
KG Koen L. van Gassen
CB Corstiaan C. Breugem
AS Alexander Stegmann
CL Caroline Lekszas
RM Reza Maroofian
EK Ehsan Ghayoor Karimiani
AB Arnaud Bruneel
NS Nathalie Seta
AM Arnold Munnich
DP Dulce Papy-Garcia
MD Muriel De La Dure-Molla
VC Valérie Cormier-Daire
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Newborn femurs were fixed in 4% paraformaldehyde, decalcified with 0.4 M EDTA before paraffin embedding and 5 μm sections were used for Safranin O and Masson’s trichrome staining or immunofluorescence.

For immunofluorescence, sections were subjected to digestion with 0.5 U/ml chondroitinase ABC (Sigma-Aldrich) for 2 h at 37 °C and, after blocking with 1% BSA, were incubated with anti-HS (1/100, Amsbio, clone F58-10E4, catalogue number 370255-1) and anti-CS (1/200, Amsbio, clone 2B6, catalogue number 270432-CS) antibodies. Alexa fluor 594 goat anti-mouse was used as secondary antibody and slides were mounted in Prolong Gold Antifade with DAPI mounting medium (1/200; Life Technologies, catalogue number A11005) and scanned using a Nanozoomer 2.0 (Hamamatsu). Specific signal intensity was measured with ImageJ software, selecting equivalent areas of the growth plate in each group.

Copyright and License information: The Author(s) ©2018
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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