Combatting Autofluorescence When Immunolabeling

Without Sudan Black bWith Sudan Black b

During immunofluorescent analysis of certain tissue structures, autofluorescence (also known as native fluorescence) can present obstacles to obtaining the highest quality microscopic images. Autofluorescence is the tendency of some tissue types to fluoresce under certain wavelengths of light. When this endogenous fluorescence occurs, there is a large amount of noise and background signal which can mask true immunolabeling and reduce the reliability and specificity of the label of interest.

Different tissue types express a myriad or different protein epitopes and in different degrees. These protein epitopes, defined by the amino acid sequence of the polypeptide, present a practically infinite range or patterns that define their fluorescent tendencies – aromatic amino acids such as tryptophan and tyrosine are especially autofluorescent. At the polypeptide level, lipoproteins like lipofuscin in central nervous system tissue are notorious for high autofluorescence – especially when exposed to higher intensity wavelengths such as 488 nm and 555 nm.

Fighting Autofluorescence

How does Visikol combat autofluorescence to obtain specificity within immunolabeling? Visikol’s experience with central nervous system tissue has found that the immunofluorescent channels 488 nm and 555 nm were so saturated with autofluorescence that distinguishing between endogenous autofluorescence, and antibody labeling, became more difficult. Conversely, the lower intensity wavelengths of 594 nm and 647 nm had little to no autofluorescence. This presented a unique challenge of how to produce a multichannel, immunofluorescent image without diluting true immunolabel signal.

Sudan Black is a common stain for histological processes and can also be used to quench particularly pesky autofluorescence. Application of 0.1% Sudan Black in 70% Ethanol over a multitude of fluorescent wavelengths can assist in solving many issues, while presenting other challenges. The channels imaged with higher intensity wavelengths (488 nm and 555 nm) saw little to no autofluorescence after the Sudan Black application. Specific immunolabeling could be seen without the very loud and intrusive glow of the autofluorescence. However, as the intensity of the wavelengths decreased, Sudan Black presented its own fluorescence – especially when imaged with a wavelength of 647 nm, masking the specific immunolabel at that intensity. Although the Sudan Black quenched the autofluorescence at the higher intensity channels (488 nm and 555 nm), it diluted the true signal at the lower intensity channels (647 nm).

This troublesome swap of nonspecific fluorescence led to more troubleshooting and clever problem solving for the issue at hand. Multiple imaging runs of strategically placed labeling within the central nervous system tissue with and without Sudan Black. Immunolabels that required higher intensity wavelengths were imaged with Sudan Black while the immunolabels which required lower intensity wavelengths were imaged without it. This allows the Image Analysis team to overlay the images with limited endogenous tissue autofluorescence over the images without Sudan Black’s unique fluorescence at a wavelength of 647 nm.

Interested in speaking to a member of our team regarding autofluorescence? Reach out today.

This troublesome swap of nonspecific fluorescence led to more troubleshooting and clever problem solving for the issue at hand.

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