An effective and practical immunohistochemical protocol for bone specimens characterized by hyaluronidase and pepsin predigestion combined with alkaline phosphatase-mediated chromogenic detection

Abstract

The aim of this study was to provide an effective procedure for immunohistochemistry (IHC) investigations of bone specimens. Samples from rat femoral and human vertebral bone were processed with a detailed and effective IHC protocol summarized here. First, a novel antigen retrieval (AR) method of hyaluronidase combined pepsin predigestion (H+P) was established and the optimal concentration and pH value for AR of bone specimens were determined. Second, the newly developed method was compared with existing AR methods (boiling in sodium citrate, hyaluronidase predigestion (H) and pepsin predigestion (P), with PBS only as the negative control) using two chromogenic detection systems (horseradish peroxidase (HRP) and alkaline phosphatase (AP)) to evaluate their efficacy in obtaining the best IHC results for bone samples. Considering the drawbacks of significant shrinking and detachment from slide for heat retrieval methods and the only moderate immunolabeling for H and P, H+P was the optimal AR method for IHC of bone specimens with the advantages of both good morphological preservation and strong immunoreactivity. Moreover, AP-mediated chromogenic detection was superior to HRP-labeled chromogenic detection due to significantly less nonspecific staining. In conclusion, we presented an effective and practical IHC protocol for bone specimens characterized by H+P predigestion combined with APmediated chromogenic detection. Finally, a detailed troubleshooting guide was provided for common mistakes that occur during IHC processing of the bone tissue samples.