Intestinal lipofuscin is particularly problematic for immunofluorescence protocols as it is inherently fluorescent. Sudan Black B (SBB) has been used successfully in the past to mask lipofuscin autofluorescence, as well as for staining of mycobacteria. Mycobacteria produce unique cell-wall lipids; therefore, SBB may not be suitable for inclusion in an immunofluorescence protocol that includes a Mycobacterium avium subspecies paratuberculosis (MAP) primary antibody. The effect of SBB, when compared to 3,3’-diaminobenzidine (DAB), on immunofluorescent antibody labeling was compared for MAP-specific antibody labeled, and TLR4- specific antibody labeled frozen bovine intestinal tissue sections. The effect of SBB on immunofluorescent antibody labeling when added prior to the primary antibody or after the secondary antibody was also compared. The masking ability of SBB was also compared with DAB for unlabeled tissue sections. When compared to DAB only treated tissue, SBB added before the primary or after the secondary antibody reduced the intensity and abundance of immunofluorescently labeled MAP. We recommend that the use of SBB be excluded from immunofluorescence protocols that utilize a MAP-specific primary antibody in order to optimize immunofluorescent labeling.
Keywords: Bovine, eosinophil, frozen, intestine, lipofuscin, Mycobacterium avium subsp, paratuberculosis, Sudan Black