Anti opn ab8448

Mouse and human lung tissues were fixed with 10% buffered formalin phosphate, embedded in paraffin, and stained with hematoxylin and eosin (H&E). The von Kossa technique was used for  staining microliths by incubation of histological sections with 3% silver nitrate, exposure to UV light for 30 min, and counterstaining with Nuclear Fast Red (Newcomer Supply; Middleton, WI). For TRAP staining, tissue sections were incubated with pre-warmed (37 °C) TRAP staining mix (University of Rochester Medical Center TRAP protocol) for 1 h and counterstaining with Harris hematoxylin for 5 sec. Immunohistochemical analysis was performed on formalin-fixed, paraffin embedded material. Five micrometer sections were deparaffinized and rehydrated with dH₂O. The sections were subjected to antigen retrieval with citrate buffer, blocked with normal serum, and probed with specific primary antibodies anti-OPN (ab8448, polyclonal, 1:7500) and anti-cathepsin K (ab19027, polyclonal, 1:750) obtained from Abcam (Cambridge, UK), anti-CALCR LS (LS-A769, polyclonal, 9ug/ml) obtained from LS Bio (Seattle, WA) followed by horseradish peroxidase linked anti-rabbit secondary antibody (Cell Signaling Technology Inc., Beverly, MA). Slides were developed with DAB substrate, counter stained with hematoxylin, dehydrated, and mounted.