How do I check for autofluorescence ?
Autofluorescence is present in many cells and tissues and has many sources including molecules of the respiratory chain like NADP/NADPH. Autofluorescence can vary greatly with the fixation method. Glutaraldehyde is in particular known for generating extremly strong autofluorescence, and therefore is not recommended for immunofluorescence experiments. Autofluorescence usually has a very wide spectrum and can be observed, i.e. if a fluorescent structure is visible with UV-, FITC and Rhodamine filter sets it is highly likely that this structure indeed is autofluorescent. autofluorescence typically is highest with UV or blue excitation and almost absent in the far red excitation. To estimate the degree of autofluorescence in an immunofluorescence experiment have control slides that are treated in the same way as your stained slides (i.e. fixation, washes, permeabilizations) but omit the florescent label (i.e. secondary antibody). Autofluorescence is often a problem in cells that are only w
Autofluorescence is present in many cells and tissues and has many sources, including molecules of the respiratory chain like NADP/NADPH. Autofluorescence can vary greatly with the fixation method. Glutaraldehyde in particular is known for generating extremely strong autofluorescence, and therefore is not recommended for immunofluorescence experiments. Autofluorescence usually has a very wide spectrum and can be observed (i.e., if a fluorescent structure is visible with UV, FITC and Rhodamine filter sets, it is highly likely that this structure is indeed autofluorescent). Autofluorescence typically is highest with UV or blue excitation and almost absent in the far-red excitation. To estimate the degree of autofluorescence in an immunofluorescence experiment, you should have control slides that are treated in the same way as your stained slides (i.e., fixation, washes, permeabilizations) but omit the flourescent label (i.e., secondary antibody). Autofluorescence is often a problem in ce
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