Anti sfrp3

Cytoplasmic extracts were prepared by homogenizing the tissues in lysis buffer as described (Wimbauer et al., 2012 (link)). The protein concentration was determined by Bradford protein assay, and cytoplasmic extracts containing protein (60 μg) were analyzed by western blot hybridization as previously shown (Benedikt et al., 2010 (link); Wimbauer et al., 2012 (link)) using anti-sFRP3 (1:2000 dilution) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000 dilution) antibodies (Santa Cruz Biotechnology, Dallas, TX). The expression levels of proteins on the western blots were quantified using densitometer and Imagelab software (BioRad, Hercules, CA).

Tissue microarrays representing malignant and normal bone were purchased from US Biomax, Inc (Rockville, MD) and analyzed by immunostaining, using anti-sFRP3 (1: 25 dilution), anti-axin2 (1: 50 dilution)and non-immune immunoglobulin (IgG) (1:200 dilution) (Santa Cruz Biotechnology, Dallas, TX). The anti-sFRP3- and anti-axin2-stained tissue arrays were normalized using IgG staining and the quantitation of signals was carried out using BIOQUANT OSTEO Image analysis system (Bioquant Image Analysis Corporation, Nashville, TN). The average densities for sFRP3 and Axin staining were calculated in normal and osteosarcoma tissues. The average density was determined by calculating the intensities of all significant pixels in the object and dividing that value by the number of pixels as described in the manufacturer’s protocol (Bioquant Image Analysis Corporation).