Quantitative measurement of neuronal calbindin-D28k by radioimmunocytochemistry.

Abstract

The calcium-binding protein calbindin-D28k (CaBP) has been localized in high concentration in several neuronal populations within the CNS and is believed to act as an intracellular calcium buffer. There has been much interest and speculation concerning its potential neuroprotective function. A radioimmunocytochemistry (RIC) technique for the cellular quantitation of protein has been applied to quantitative measurement of neuronal CaBP in vivo and in vitro. The method permits cellular comparison of CaBP content within tissue sections or cells in culture. Through the use of specific primary antibody, 35S-labeled secondary antibody, and photographic emulsion, RIC combines the simplicity of standard immunocytochemical procedures with the sophistication and power of in situ hybridization, autoradiography, and image analysis. CaBP levels are expressed as mean +/- S.E.M. silver grains/cell. CaBP content has been measured and compared in mouse cerebellar Purkinje cells (56.5 +/- 6.9 grains/cell), granule cells of the hippocampal dentate gyrus (10.3 +/- 2.1 grains/cell), midline ventral tegmental neurons (11.6 +/- 2.9 grains/cell), and human SH-SY-5Y neuroblastoma cells in culture (5.1 +/- 0.9 grains/cell). As measured by RIC, mouse cerebellar Purkinje cells contain approximately 5-fold more CaBP than granule cells of the hippocampal dentate gyrus/midline ventral tegmental neurons and 10-fold more CaBP than cultured human SH-SY-5Y neuroblastoma cells. Assay reproducibility was demonstrated by comparison of adjacent sections which yielded a 3-9% intra-assay variability. Results were validated and confirmed by comparison to previous radioimmunoassay studies which indicated similar ratios of CaBP levels between brain regions/cell types.(ABSTRACT TRUNCATED AT 250 WORDS)