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Journal of Histochemistry and Cytochemistry, Vol. 47, 411-420, March 1999, Copyright © 1999, The Histochemical Society, Inc.

TECHNICAL NOTE

A Semiquantitative Image-analytical Method for the Recording of Dose–Response Curves in Immunocytochemical Preparations

Helmut Wichta, Erik Marondea, James Olceseb, and Horst-Werner Korfa
a Dr. Senckenbergische Anatomie, Anatomisches Institut II, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt, Germany
b Institut für Hormon- und Fortpflanzungsforschung, Universität Hamburg, Hamburg, Germany

Correspondence to: Helmut Wicht, Anatomisches Institut II, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt, Germany. email: wicht@em. uni-frankfurt.de

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose–response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to ß-adrenergic stimuli inducing 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 CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose–response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose–response curves from 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 quantification and localization of biomolecules, and may even be more sensitive than immunoblotting. (J Histochem Cytochem 47:411–419, 1999)

Key Words: immunocytochemistry, cell cultures, image analysis, quantification, cytopharmacology, third messengers, phosphorylation


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