1x70 inverted microscope

Following fixation in 10% paraformal-dehyde for 48 h, the rat femoral head was decalcified with 10% EDTA (pH 7.2) for ~42 days, with refreshing once/week until no significant resistance to pin passing through. Following washing with running water for 24 h, the decalcified femur head was dehydrated with upward gradient alcohol solution, transparented with xylene, embedded in paraffin, sliced to 5 mm and mounted onto poly-lysine-coated slides. The tissue sections were stained for Wnt3a and β-catenin using 100-fold diluted primary antibodies provided in the SABC kit in strict accordance with the manufacturer's protocol. A negative control was performed by replacing primary antibodies with PBS buffer. A total of five randomly selected fields (magnifi-cation, x400) were observed and images were captured using an Olympus 1X70 inverted microscope. Wnt3a and β-catenin positive cells, which were defined by the appearance of a brown substance in the cell membrane, cytoplasm and nucleus, were counted, and their gray values were measured using MOTIC6.0 software (Motic China). The average gray value was used to represent staining intensity.