Alzheimer's disease (AD), the most prevalent dementia linked to aging, involves neurotoxic effects of amyloid β species and dishomeostasis of intracellular Ca2+. To investigate mechanisms of AD, the effects of soluble species of amyloid β oligomers (Aβo) prepared in medium devoid of glutamate receptor agonists can be tested on intracellular Ca2+ in long-term cultures of rat hippocampal neurons that reflect aging neurons. Furthermore, changes in expression of proteins involved in oligomer responses and AD can be tested in the same neurons using quantitative immunofluorescence. Detailed procedures for the preparation of Aβ species in defined medium, long-term culture of rat hippocampal neurons mimicking aged neurons, calcium imaging and quantitative immunofluorescence in these cultures are described in this chapter.