sepharose and deoxyuridine-triphosphate

Organotypic brain slices (OTBS) are an excellent experimental compromise between the facility of working with cell cultures and the biological relevance of using animal models where anatomical, morphological, and cellular function of specific brain regions can be maintained. The biological characteristics of OTBS can subsequently be examined under well-defined conditions. They do, however, have a number of limitations; most brain slices are derived from neonatal animals, as it is difficult to properly prepare and maintain adult OTBS. There are ample problems with tissue integrity as OTBS are delicate and frequently become damaged during the preparative stages. Notwithstanding these obstacles, the introduced exogenous proteins into both neuronal cells, and cells imbedded within tissues, have been consistently difficult to achieve.. Following the ex vivo extraction of adult mouse brains, mounted inside a medium-agarose matrix, we have exploited a precise slicing procedure using a custom built vibroslicer. To transfect these slices we used an improved biolistic transfection method using a custom made low-pressure barrel and novel DNA-coated nanoparticles (40 nm), which are drastically smaller than traditional microparticles. These nanoparticles also minimize tissue damage as seen by a significant reduction in lactate dehydrogenase activity as well as propidium iodide (PI) and dUTP labelling compared to larger traditional gold particles used on these OTBS. Furthermore, following EYFP exogene delivery by gene gun, the 40 nm treated OTBS displayed a significantly larger number of viable NeuN and EYFP positive cells. These OTBS expressed the exogenous proteins for many weeks.. Our described methodology of producing OTBS, which results in better reproducibility with less tissue damage, permits the exploitation of mature fully formed adult brains for advanced neurobiological studies. The novel 40 nm particles are ideal for the viable biolistic transfection of OTBS by reducing tissue stress while maintaining long term exogene expression.

Topics: Animals; Bacterial Proteins; Biolistics; Brain; Brain Chemistry; Deoxyuracil Nucleotides; DNA; DNA-Binding Proteins; Gene Expression; Gene Transfer Techniques; L-Lactate Dehydrogenase; Luminescent Proteins; Mice; Mice, Inbred C57BL; Microtomy; Nanoparticles; Nerve Tissue Proteins; Nuclear Proteins; Propidium; Sepharose; Staining and Labeling; Transgenes

Sites of DNA replication in nuclei are focally concentrated, suggesting that an underlying structure organizes the activity of many polymerases. As fixation could induce aggregation into foci, we examined the distribution of replication sites in unfixed nuclei. HeLa cells were encapsulated in agarose microbeads, permeabilized in a 'physiological' buffer, their DNA polymerizing activity characterized, and replication sites directly labelled by incubation with fluorochrome-dUTP conjugates. Using conventional and digital fluorescence microscopy, 80-250 foci were seen in these unfixed cells. These foci are unlikely to be formed by the aggregation of separate polymerases as most replication activity found in vivo is retained throughout these procedures. Although commonly used fixation methods collapsed or dispersed their periphery, the central core was very stable. Foci remained when approximately 90% chromatin was removed, suggesting they were attached to an underlying structure.

Topics: Artifacts; Cell Membrane Permeability; Deoxyuracil Nucleotides; DNA Replication; DNA-Directed DNA Polymerase; Drug Compounding; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Microspheres; Nuclear Envelope; Nuclear Matrix; Sepharose