A semiquantitative image-analytical method for the recording of dose-response curves in immunocytochemical preparations

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli a re typically quantified via RIA, ELISA, or immunoblots. These techniques, w hich require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the mole cules under investigation. We describe a protocol for recording dose-respon se curves directly from immunocytochemical preparations using rat pinealocy tes as a model system. The cells were exposed to p-adrenergic stimuli induc ing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin . Melatonin concentrations were determined by ELISA. cPKA, phosphorylated C REB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dos e-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves f rom immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quan tification and localization of biomolecules, and may even be more sensitive than immunoblotting.