Biotinylated sheep anti mouse igg

Levels of intercellular BMP-4 and that in conditioned medium were determined by ELISA using mouse anti-human BMP-4 (R&D) as the primary antibody, with biotinylated sheep anti-mouse IgG (Amersham) and avidin-HRP (Boehringer) utilized as secondary antibodies.

Immuno-histochemistry was performed on frozen sections stained with the primary antibodies for a-SMA (clone 1A4; Dako, Glostrup, Denmark), CD68 (clone KP1, Dako), uncarboxylated and carboxylated MGP (ucMGP and cMGP, respectively; 1:25; IDS, Boldon, UK), BMP-2 (1:20; Genetics Institute, Cambridge, MA) and Osteocalcin (1:50; Anawa Trading, Wangen, Zürich, Switzerland). Secondary antibodies used were Biotinylated sheep anti-mouse IgG (1:250; Amersham, Little Chalfont, Buckinghamshire, UK) or sheep anti-rabbit IgG (1:1000, Dako). Antibodies were visualized by alkaline phosphatase–coupled avidin-biotin complex (Dako), in combination with red alkaline substrate kit I (Vector SK-5100; Vector Laboratories, Burlingame, CA); nuclei were counterstained with hematoxylin. Furthermore, all samples we routinely stained for Hematoxylin Eosin (HE), von Kossa, oil red O and Picro-Sirius red.