A standard indirect IHC procedure was used to detect IGF-1, BMPR1b and MMP-10 expression (Dobie et al., 2015 (link)). Primary antibodies were anti-IGF-1 (Abcam, ab9572, 1:500 dilution), BMPR1b (Abcam, ab175385, 1:100 dilution) and MMP-10 (Abcam, ab199688, 1:100 dilution). Control sections were incubated with an equal amount of rabbit IgG in place of the primary antibody. Sections were counterstained by Haematoxylin, mounted and images captured using a Zeiss AxioImager brightfield microscope. Fixation and antigen retrieval were carried out using the same methodology throughout and all IHC for each antibody was performed on the same day under identical conditions, with control specimens also tested for each genotype. After the images were imported into Fiji, the Haematoxylin-DAB colour deconvolution plugin was used to separate the Haematoxylin and DAB components, and the ‘analyse-measure’ tool used to determine the absorbance in a consistent region of each sample, containing both GP and metaphyseal bone. Optical density (OD) was then calculated using the equation: OD=negative (base10)log of mean intensity of transmitted image/illumination (max intensity of image).
Maximum intensity was taken to be 255 for 8-bit images in Fiji (Ruifrok and Johnston, 2001 (link)).
Maximum intensity was taken to be 255 for 8-bit images in Fiji (Ruifrok and Johnston, 2001 (link)).