In hippocampal neurons, certain mRNAs have been found in dendrites (Steward, 1997), and their localization and translation have been implicated in synaptic plasticity (Martin et al., 1997). One attractive candidate to achieve transport of mRNAs into dendrites is Staufen (Stau), a double-stranded RNA-binding protein, which plays a pivotal role in mRNA transport, localization, and translation in Drosophila (St. Johnston, 1995). Using antibodies raised against a peptide located in the RNA-binding domain IIa and a polyclonal antibody raised against a recently cloned human Staufen homolog, we identify a 65 kDa rat homolog in cultured rat hippocampal neurons. In agreement with the exclusive somatodendritic localization of mRNAs in these cells, we find that Staufen is restricted to the same domain. By immunoelectron microscopy, we show enrichment of the mammalian homolog of Stau (mStau) in the vicinity of smooth endoplasmic reticulum and microtubules near synaptic contacts. Finally, the association of the mStau with neuronal mRNAs is suggested by the colocalization with ribonucleoprotein particles specifically in distal dendrites known to contain mRNA, ribosomes, and translation factors (Knowles et al., 1996). These results suggest a role for mStau in the polarized transport and localization of mRNAs in mammalian neurons.
Transfection of primary neurons in culture has proven to be experimentally challenging in the past. To overcome these limitations, we present a detailed transfection protocol for hippocampal neurons based on DNA/Ca(2+)-phosphate coprecipitation. The main advantages being (1) the speed and convenience, (2) the remarkable efficiency of transfection for mature neurons, and (3) consistent health of the neurons upon transfection allowing subsequent manipulations. The strength of this protocol is convincingly demonstrated by the fact that the expressed protein can be detected biochemically on Western blots.
In stem-cell research a major difficulty is caused by the lack of distinctive features that allow the identification of human mesenchymal stem cells (hMSC). Until now, there has been no specific marker and the most common way to identify hMSC is by their characteristic stem-cell properties: self-replication and differentiation potential.However, these findings can only be revealed retrospectively, and, once differentiated, hMSC lose their stem-cell character. The aim of this study was to establish four-colour immunofluorescence of several markers simultaneously in order to address the problem of how to identify hMSC on the single-cell level. The four markers collagen-I, collagen-IV, fibronectin and CD44 are known to be expressed by hMSC. Antibody binding was detected using secondary antibodies conjugated to FITC, Alexa546, TexasRed and AMCA. Because the distinction between Alexa546 and TexasRed was not possible on conventional digital images using standard filter sets, we performed spectral image acquisition. The image was subsequently decomposed into its pure spectral components, which permitted linear unmixing. Using this procedure we were able to demonstrate four-colour immunofluorescence on hMSC. With the possibility of using more sophisticated marker profiles and / or additional markers, four-colour immunofluorescence offers the opportunity of identifying hMSC on the single-cell level without performing differentiation assays.
Transfection of primary neurons in culture has proven to be experimentally challenging in the past. To overcome these limitations, we present a detailed transfection protocol for hippocampal neurons based on DNA/Ca(2+)-phosphate coprecipitation. The main advantages being (1) the speed and convenience, (2) the remarkable efficiency of transfection for mature neurons, and (3) consistent health of the neurons upon transfection allowing subsequent manipulations. The strength of this protocol is convincingly demonstrated by the fact that the expressed protein can be detected biochemically on Western blots.
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