Multicolor fluorescence immunohistochemistry of human prefrontal cortex

Abstract

Multicolor fluorescence immunohistochemistry on human postmortal brain samples was optimized to allow simultaneous imaging of more than five markers on one section. The main difficulty of fluorescence immunohistochemistry on human nervous tissue is its high background autofluorescence due to formaldehyde fixation and lipofuscin glycolipoprotein fluorescence. We aimed to minimize background autofluorescence and, simultaneously, enhance signal from molecular markers of the proteins of interest. Here we present the results of multicolor fluorescence immunohistochemistry on 30 mkm sections of human prefrontal cortex stained with antibodies against AQP1, CHRNB3, GFAP and cellular nuclei and blood vessels imaging. We demonstrated, that mild formaldehyde fixation and treatment with 1% Sudan black B in 70% ethanol together with blocking steps, preventing unspecific binding of antibodies, streptavidin conjugates and AlexaFluor, decreased background fluorescence, and application of secondary / tertiary detection systems with AlexaFluor / streptavidin conjugates enhanced specific immunohistochemical signal.