Autofluorescence In Sections Research Articles

Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

Effect of tissue-processing techniques on the sensitivity of microfluorimetric determinations of tracers in tissue sections

The measurement of low concentrations of fluorescent tracers in sections of tissue is theoretically possible using several microfluorimetric techniques but the sensitivity attainable can be greatly decreased if high levels of autofluorescence are emitted by tissue components. The effects of tissue-processing techniques on autofluorescence and tracer fluorescence intensities were investigated. Autofluorescence in sections of rabbit carotid artery and fluorescence from lissamine rhodamine B-labelled albumin in a model system were measured by densitometry. Autofluorescence intensities appeared to be related to the degree of cross-linking resulting from fixation of the tissue. Tissue fixed with formaldehyde or mercury (II) chloride had the lowest emission. Fluorescence from the labelled albumin was not quenched by prolonged fixation with mercury (II) chloride and formaldehyde but extracting the “mercury pigment” from sections with alcoholic iodine resulted in a 34% decrease in intensity.

Immunofluorescence studies with 4-acetamido-4′-isothio-cyanato stilbene-2,2′-disulphonic acid (SITS)

The application of the fluorochrome 4-acetamido-4∝isothiocyanato stilbene-2,2∝-disulphonic acid (SITS) in immunofluorescence was studied. The optimal excitation wave length is 350 nm, and optimal fluorescence is obtained at 420 nm. After purification of the commercial compound, conjugation is performed in a strong buffer at pH 9.0–9.5. SITS conjugates were very satisfactory for immunofluorescence studies of the cytoplasmic antigens of cell preparations, but their blue emission was difficult to distinguish from autofluorescence in sections of human tissues. Good results with immunofluorescence on membrane bound antigens were obtained by using an ultra-violet laser beam as light source. SITS can be used simultaneously with FITC and TRITC conjugates thus making it possible to show three antigens in one preparation.