Rabbit anti human alp

Immunohistochemical staining was performed as previously described.21 Paraffin serial sections (4 μm) were obtained, and immunohistochemical evaluations were performed using the following primary antibodies: mouse anti‐human α7 nAChR (1:1000; BioLegend), rabbit anti‐human ALP (1:1000; Abcam), rabbit anti‐human RUNX2 (1:200; Santa Cruz), mouse anti‐human GSK‐3β (1:250; Cell Signaling) and mouse anti‐human phosphorylated GSK‐3β (1:50; Cell Signaling). Biotin‐labelled secondary antibodies, including goat antimouse and goat anti‐rabbit antibodies (ZSGB‐bio), were used. Measurements of integrated optical density were conducted using Image Pro Plus version 6.0 software (Azure). Representative results from one of three independent experiments are shown.

Western blot was performed according to a previously described protocol.22 The primary antibodies used in this study included rabbit anti‐human ALP (1:2000; Abcam), rabbit anti‐human RUNX2 (1:500; Santa Cruz), mouse anti‐human BSP (1:500; Santa Cruz), mouse anti‐human OCN (1:500; Santa Cruz), mouse anti‐human RANKL (1:500; Santa Cruz), rabbit anti‐human OPG (1:500; Santa Cruz), mouse anti‐human α7 nAChR (1:1000; BioLegend), mouse anti‐human GSK‐3β (1:1000; Cell Signaling), rabbit anti‐human phosphorylation GSK‐3β (1:1000; Cell Signaling) and mouse anti‐human β‐actin (1:2000; Comwin). Horseradish peroxidase‐conjugated goat anti‐rabbit and goat antimouse secondary antibodies (1:5000; Jackson) were used. β‐actin served as the internal reference, and semi‐quantitation of protein expression was conducted by determining the intensity of the targeted protein bands using densitometry and Photoshop CS6 (Adobe Systems). Representative results from one of three independent experiments are shown.