Streptavidin biotinylated horseradish peroxidase complex

Immunohistochemical detection of eGFP and MKP‐1 expression in the lungs was performed with the immunoperoxidase method using the streptavidin‐biotinylated horseradish peroxidase complex (DAKO), as described in a previous study.³²

Immunohistochemical staining was carried out according to the manufacturer’s instructions. All sections were dewaxed, soaked in ethanol, and treated with 3% hydrogen peroxide to block endogenous peroxidase activity. Nonspecific immunoreactivity was blocked by incubating the sections in normal rabbit serum at room temperature. The tumor tissue sections were then incubated with primary monoclonal antibodies CD34, CK19, Ki67, AFP, Glypican-3 (working solution, Gene Tech Company limited). Then, secondary biotinylated antimouse immunoglobulin (Dako, Denmark) was applied and then reacted with streptavidin biotinylated horseradish peroxidase complex (Dako, Denmark). The sections were stained with diaminobenzidine solution and then counterstained with Mayer’s hematoxylin. The negative control was obtained by substituting the primary antibodies with mouse immunoglobulin G. The nuclear fraction of Ki67 positivity was quantitatively measured as previously described.11 (link) All patients were divided into positive and negative Ki67 groups based on the median value. Expression of CD34, AFP, GPC3 with moderated or strong staining in tumor cells was defined as positive expression.11 (link)