tetramethylrhodamine and cyanine-dye-3

Topics: Base Sequence; Biomedical Enhancement; Biosensing Techniques; Carbocyanines; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gold; Metal Nanoparticles; Models, Molecular; Rhodamines; Surface Plasmon Resonance; Surface Properties

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Topics: Animals; Carbocyanines; Cell Separation; Computer Systems; Corpus Striatum; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Frozen Sections; Hydrazines; Interneurons; Isoenzymes; Lasers; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Rhodamines; RNA, Messenger; Specimen Handling; Time Factors

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Topics: Antibodies, Monoclonal; Calcium; Carbocyanines; Carcinoma; Cell Membrane; Dimerization; Energy Transfer; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Intracellular Fluid; Microscopy, Fluorescence; Phosphorylation; Rhodamines; Signal Transduction; Tumor Cells, Cultured