A11-positive ß-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis.

ß-amyloid (Aß) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, Aß oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer's disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, Aß oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of Aß oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen Aß1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic Aß1-42 oligomer with anti-parallel ß-sheet structures. In this protocol, we describe how to prepare an A11-positive Aß1-42 oligomer-rich sample from a synthetic Aß1-42 peptide in vitro and to evaluate relative amounts of A11-positive Aß1-42 oligomer in samples by a dot blotting analysis using A11 and Aß1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive Aß1-42 oligomer can also be screened from semi-quantitative experimental results.

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.