Bone samples were fixed overnight in either 95% ethanol/5% acetic acid or in ice-cold 4% PFA. They were then decalcified in 0.25 M EDTA, embedded in paraffin wax and sectioned sagittally and mounted on positively charged glass slides. For haemotoxylin and eosin (H & E) staining, the slides were dewaxed in xylene, rehydrated and H and E stained using a ThermoShandon Ltd automated stainer, dehydrated in an ethanol gradient, cleared in xylene and mounted using a xylene-based mounting solution.
Hypertrophic zone width measurements were taken in the central part of the image. The start of the zone was defined as the point at which the disc shape cells of the proliferative zone started to round up and become larger, and the end of the zone taken as the vascular invasion front. Measurements were performed using the Photoshop Ruler Tool on images of known magnification. For each animal, three separate sections spaced at least 75 µm apart were averaged.
Immunohistochemistry against collagen X was carried out on ethanol/acetic acid-fixed samples. A rabbit polyclonal antibody to recombinant mouse collagen X NC1 domain was produced (Eurogentec). Briefly, a Antibodies to BrdU, and the myc epitope (Roche -) were used on PFA fixed tissue as described [48] (link). BrdU labelling was carried on 3 week old mice which were injected intraperitoneally with 100 mg BrdU (Sigma) per kg body weight, and sacrificed 2 hours later. Tibias were fixed in ice-cold 4% PFA, demineralised in 4% PFA containing 0.25 M EDTA and sectioned as described above. Antigen unmasking was performed in 4 M HCl for 15 minutes, neutralised with 0.1 M borate buffer. The number of BrdU-labelled cells was expressed as a proportion of the total population of cells in the proliferative zone which was defined as the region of the growth plate in which chondrocytes are disc shaped and form columns. Images were prepared using a Carl Zeiss Axiocam Colour CCD camera with associated Axiovision software and processed using Adobe Photoshop.
Hypertrophic zone width measurements were taken in the central part of the image. The start of the zone was defined as the point at which the disc shape cells of the proliferative zone started to round up and become larger, and the end of the zone taken as the vascular invasion front. Measurements were performed using the Photoshop Ruler Tool on images of known magnification. For each animal, three separate sections spaced at least 75 µm apart were averaged.
Immunohistochemistry against collagen X was carried out on ethanol/acetic acid-fixed samples. A rabbit polyclonal antibody to recombinant mouse collagen X NC1 domain was produced (Eurogentec). Briefly, a Antibodies to BrdU, and the myc epitope (Roche -) were used on PFA fixed tissue as described [48] (link). BrdU labelling was carried on 3 week old mice which were injected intraperitoneally with 100 mg BrdU (Sigma) per kg body weight, and sacrificed 2 hours later. Tibias were fixed in ice-cold 4% PFA, demineralised in 4% PFA containing 0.25 M EDTA and sectioned as described above. Antigen unmasking was performed in 4 M HCl for 15 minutes, neutralised with 0.1 M borate buffer. The number of BrdU-labelled cells was expressed as a proportion of the total population of cells in the proliferative zone which was defined as the region of the growth plate in which chondrocytes are disc shaped and form columns. Images were prepared using a Carl Zeiss Axiocam Colour CCD camera with associated Axiovision software and processed using Adobe Photoshop.
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