IMMUNOCYTOCHEMICAL DETECTION OF REGIONAL PROTEIN-CHANGES IN RAT-BRAINSECTIONS USING COMPUTER-ASSISTED IMAGE-ANALYSIS

We used several approaches to assess the reliability and sensitivity o f computer-assisted densitometry to detect regional changes in tissue antigen content as a function of immunohistochemical staining density. We designed a model system to mimic variations in antigen concentrati on in postfixed, slide-mounted rat brain sections by varying the ratio s of conjugated (biotinylated) to unconjugated secondary antibody, Ant igen concentration was also varied in tissue discs made from mixing ra t brain homogenate with increasing amounts of tissue embedding compoun d. The monoclonal antibody bd-17 to the beta(2/3) subunit of the GABA( A) receptor was used as the primary antibody, Immunostaining density w as visualized with diaminobenzidine (DAB). There was a significant, po sitive linear relationship (r = 0.97-0.99) between immunostaining inte nsity and antigen concentration. With this approach, changes in antige n content of less than 10%, as reflected in immunostaining intensity, were detectable in brain sections. The low degree of variability in me asures of regional variation in immunostaining in sections from naive rats (n = 7) suggested that the method was suitable for quantitative a nalysis and indicated the reliability of the method. This systematic s tudy of the utility of computer-assisted image analysis for semiquanti tative immunohistochemical analysis found the method to be both reliab le and sensitive.