R d hrp dab staining kit

Paraffin sections in 5 µm were used. Osteoclast was carried out using a standard protocol by TRAP (tartrate-resistant acid phosphatase) staining (Sigma–Aldrich). Immunohistochemical staining was achieved by applying a standard protocol with the R&D HRP-DAB staining kit (R&D Systems, USA). Primary antibodies matrix metallopeptidase 13 (MMP13, Abcam, Cambridge, UK; 1:200, ab3208), osterix (OSX) (Abcam, 1:200, ab22552), osteocalcin (OCN) (Takara Bio Inc., Shiga, Japan; 1:200, M137); a TGFβ pathway-specific antibody against p-Smad2/3 (Santa Cruz Biotechnology, 1:100, sc-11769); and antibody against PTH1R (Abcam, 1:200, ab15750). A biotinylated secondary antibody was applied, and then all sections were counterstained with hematoxylin. Image J software evaluated the numbers of positive cells.

Paraffin-embedded samples were sectioned at a thickness of 5 μm. Selected sections were then stained with haematoxylin and eosin (H&E) and analysed under bright-field microscopy (BX53; Olympus, Tokyo, Japan). For Masson's trichrome staining, we used Weigert's iron haematoxylin, Biebrich scarlet-acid fuchsin and aniline blue. Immunohistochemical analyses were performed on selected sections of each construct using a tooth-specific antibody against amelogenin (sc-32892; Santa Cruz Biotechnology, Paso Robles, CA, USA), a TGF-β pathway-specific antibody against p-Smad2/3 (sc-11769; Santa Cruz Biotechnology, Paso Robles, CA, USA) and an antibody against smad2/3 (NBP1-19520; Novus Biologicals, Littleton, CO, USA). For immunohistochemistry (IHC), all sections were incubated with a biotinylated secondary antibody, stained using the R&D HRP-DAB staining kit (R&D Systems, Minneapolis, MN, USA) and counterstained with haematoxylin. Processed sections were dehydrated through a series of graded ethanol baths, sealed with Permount (Thermo Fisher Scientific, Waltham, MA, USA), and analysed using bright-field microscopy. Photographs were obtained with a digital camera and manipulated using Adobe Photoshop.