Sudan Black B masks Mycobacterium avium subspecies paratuberculosis immunofluorescent antibody labeling

Authors: Stabel, Judith; JENVEY, CAITLIN - US Department Of Agriculture (USDA)

Submitted to: Histology and Histopathy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2017
Publication Date: 10/15/2017
Citation: Stabel, J.R., Jenvey, C.J. 2017. Sudan Black B masks Mycobacterium avium subspecies paratuberculosis immunofluorescent antibody labeling. Histology and Histopathy. 4(11). doi:10.7243/2055-091X-4-11.
DOI: https://doi.org/10.7243/2055-091X-4-11

Interpretive Summary: Confocal microscopy is a widely used method utilizing fluorescence to identify the presence of cell types within tissues and/or bacterial or viral pathogens. This method has been used successfully to detect the presence of bacterial pathogens such as mycobacteria and to correlate that presence with the pathogenesis of disease. Understanding the pathogenesis of the disease and the host immune response to infection will allow us to develop improved diagnostic tools and vaccines. However, it is important to clearly define specific staining to discount potential false positive results caused by autofluorescence. Autofluoresence can be high, depending upon the tissue type and the source of the tissue (animal species). Ancillary stains were used to quench autofluoresence during confocal microscopy of bovine intestinal tissues from cows naturally infected with Mycobacterium avium subsp. paratuberculosis. Our results demonstrated a clear interference of the stain, Sudan Black B, with specific staining for the mycobacterium. This information will be helpful to scientists who undertake this type of methodology in their research, particularly those who work with cattle. Confocal microscopy can be used to improve existing knowledge of disease, leading to improved diagnostic tools.

Technical Abstract: Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immunofluorescence protocol development, autofluorescence and non-specific immunofluorescent labeling was observed in frozen bovine intestinal tissue from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP). Various components of the protocol were evaluated to reduce the autofluorescence such as fixing medium, serum blocking, histochemical stains Sudan Black B (SBB) and 3,3’-diaminobenzdine, and spectral restriction and unmixing were compared for their ability to reduce autofluorescence, as well as their effect (if any) on immunofluorescent labeling. During our methods optimization it was discovered that SBB also reduced specific immunofluorescent labeling and increased background autofluorescence in far-red channels (640nm). The order of use of SBB was critical, as SBB could occupy sites for antibody binding if used to pre-treat the tissues. Based upon these observations, we recommend that SBB be excluded from immunofluorescence protocols that utilize a MAP-specific primary antibody in order to optimize immunofluorescent labeling.