Intranuclear inclusions present in the brains of patients with Fragile X-associated tremor/ataxia syndrome (FXTAS) have historically been difficult to study due to their location and scarcity. The recent finding that these particles autofluoresce has complicated the use of immunofluorescence techniques, but also offers new opportunities for purification. We have ascertained the features of the autofluorescence, including its excitation/emission spectrum, similarities and differences compared with lipofuscin autofluorescence, and its presence/absence under various fixation, mounting and UV light exposure conditions. Immunofluorescence at various wavelengths was conducted to determine which conditions are ideal for minimizing autofluorescence confounds. We also present a technique for autofluorescence-based sorting of FXTAS inclusions using flow cytometry, which will allow researchers in the field to purify inclusions more successfully for unbiased analyses.
The intranuclear inclusions of FXTAS are autofluorescent across a broad range of wavelengths. We performed experiments using fluorescence microscopy to define the characteristics of this autofluorescence and to determine what immunofluorescence conditions might overcome interference from inclusion autofluorescence. The inclusion-associated autofluorescence pattern was also used to develop a method for purifying inclusions through nuclear isolation, sucrose fractionation and FACS.