Comparative image analysis of EGF immunoreaction in rat submandibular gland using 3,3'-diaminobenzidine with metal enhancer substrate.

We have shown the efficacy of image analysis using 3,3'-diaminobenzidine (DAB) with a metal enhancer substrate for demonstrating quantitative differences in the amount of epidermal growth factor (EGF) in the submandibular gland from normal, castrated, and testosterone propionate (TP) treated castrated rats. Immunohistochemical determination of EGF visualized by DAB-nickel reagent was performed with image analysis using a computed image analyzer system (ACAS 570). Immunohistochemistry for EGF disclosed positive staining in granular convoluted tubule cells in the tissue sections from each experimental group. Using the tools of a competent data program installed in the ACAS 570 software, we measured quantitative differences among the experimental glands examined. Castration was shown to elicit a significant reduction in the EGF-positive area and staining intensity, and administration of TP to the castrated animals restored these parameters to levels greater than those of normal rats. Our study demonstrates that a simple, inexpensive, commercially available metal enhancer substrate can be applied accurately to the computer assisted quantification of histochemical hormone-induction studies.