Astrocyte culture on nitrocellulose membranes and plastic: Detection of cytoskeletal proteins and mRNAs by immunocytochemistry and in situ hybridization

Abstract

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13‐mm diameter Millipore^TM disk) and on plastic coverslips in serum‐supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.