Elimination of Autofluorescence in Immunofluorescence

Elimination of autofluorescence in immunofluorescence microscopy with digital image processing.

Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluorescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have narrow spectra. Two images are recorded and digitized, one at a wavelength exciting both the fluor...

Elimination of Autofluorescence in Immunofluorescence Microscopy with Digital Image Processing1

An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues.

Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be u...

Reducing Tissue Autofluorescence

Introduction Immunofluorescence is a common technique in modern biology. It is a highly sensitive method used for the analysis of tissue samples with a fluorescence microscope. In this technique, antibodies are labeled with a fluorescent molecule in order to localize the antigen-antibody complex. Immunofluorescence (IF) can provide clear subcellular visualization of proteins, glycans, and small...

Autofluorescence generation and elimination: a lesson from glutaraldehyde.

Glutaraldehyde causes especially high autofluorescence. It reacted with proteins and peptides to generate visible to near-IR emitters. A model indicated that ethylenediamine and a secondary amine in the molecule were key components for the formation of emissive species. The mechanism enables us to control the generation and elimination of autofluorescence.