Sc 25455

BMPRIB expression was examined by histochemistry techniques and S-P method. In brief, Sections (5μm thick) were dewaxed, hydrated, and heated for 2.5 min for antigen retrieval in a conventional pressure cooker by using citrate buffer, pH 6.0. Then sections were treated with 3% H₂O₂ for 20 min to reduce endogenous activity and incubated with 10% normal goat serum for 20 min to eliminate nonspecific staining. Next, the primary antibody against BMPRIB (rabbit anti-human polyclonal antibody, Santa Cruz, USA, sc-25455, 1:100) was applied at 4°C overnight. After washing, biotin labeled secondary antibody against rabbit immunoglobulin was applied for 20 min at room temperature. The slides were rinsed and covered with streptavidin-biotin-peroxidase for 20 min. All sections were stained with 3,3′-diaminobenzidinetetra-hydrochloride (DAB). Slides were counterstained with hematoxylin and mounted for light microscopy.

Paraffin-embedded P1 lower jaws (n = 5) were sliced to a thickness of 8 μm. Selected sections were then soaked in L.A.B. Solution (Polysciences, PA, USA) for 5 min for antigen activation, followed by incubation in 5% BSA/PBS for 1 h. After incubation with the primary antibodies for BMPRIA (sc-20736; Santa Cruz Biotechnology, CA, USA), BMPRIB (sc-25455; Santa Cruz Biotechnology), or BMPRII (MABD171; Merck Millipore, MA, USA), expression was detected using Alexa488- or Alexa594-conjugated secondary antibodies (Life Technologies). A fluorescence microscope (BZ-8000, KEYENCE Co, Osaka, Japan) was used for image analysis. Images were prepared using a BZ analyzer (KEYENCE) and Photoshop (Adobe Systems, CA, USA).