Autofluorescent compounds present in intestinal tissue often hinder the ability to utilize multiple, spectrally different, fluorophores. In addition, fixatives and blocking solutions may contribute to background autofluorescence or nonspecific immunofluorescent labeling. During immunofluorescence protocol development, autofluorescent pigments were observed in frozen bovine mid-ileal intestinal tissue sections. Coagulant fixatives, normal serum blocking, histochemical stains Sudan Black B (SBB) and 3,3'-diaminobenzidine (DAB), and spectral separation using imaging software were compared for their ability to reduce autofluorescence, as well as their effect on immunofluorescent labeling. Fluorescent pigments of frozen bovine mid-ileal intestinal tissue sections, most likely caused by eosinophils and lipofuscin, were masked successfully with a combination of DAB and SBB. Little to no statistical differences were observed for all other methods investigated; however, tissue fixed with 1:1 acetone methanol and 10% horse serum diluted in 0.05 M Tris buffer demonstrated lower mean fluorescence intensities. Spectral separation of specific immunofluorescent labeling from background autofluorescence is a simple method for removing unwanted fluorescence; however, successful separation is dependent on tissue and labeling quality.