Optimising of Immunohistochemical Protocol for Organotypic Brain Slice Cultures

Abstract

Organotypic slice cultures of brain tissue is an ex-vivo technique that been used increasingly to study neuronal death induced by hypoxia, hypoglycaemia, oxidative stress and brain disorders such as; Alzheimer and Parkinson’s disease. Most of previous studies have used propidium iodide as a marker for dead or dying cells. This study aimed to evaluate existing immunohistochemical methods, in order to improve detection of organotypic slice culture of postnatal mice at 4-5 days of age, then fluorescence labelling conducted and examined by a confocal microscope. In the current study, Glial Fibrillary Acidic Protein (GFAP) dye was used for astrocyte detection instead of propidium iodide. The organic slice culture is very sensitive when placing the coverslip, so, the thickness of the mounted slices was optimized with accurate casket bridge. It was shown that a bridge mounting method after overnight incubation at 4OC in 1% Triton X- 100 in PBS provided high quality for the immunohistochemical technique. Various detergent concentrations of permeabilization were examined as well. Results are reflecting clear morphology of astrocytes and the accuracy of the permeabilization concentration.