Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections
V.C. Oliveira1, R.C.V. Carrara2, D.L.C. Simoes1, F.P. Saggioro1, C.G. Carlotti3 Jr, D.T. Covas2 and L. Neder1
1Department of Pathology, 2Center for Cell Therapy and Regional Blood Center and 3Department of Surgery, Ribeirão Preto School of Medicine University of São Paulo (USP), Ribeirão Preto - SP, Brazil.
Offprint requests to: Viviane de Cássia Oliveira Laboratório de Neuropatologia, Departamento de Patologia - Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (FMRP-USP), Av. Bandeirantes, 3900 - Ribeirão Preto SP, Brasil - 14049-900. e-mail: firstname.lastname@example.org
Summary. Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels. Histol Histopathol 25, 1017-1024 (2010)
Key words: Autofluorescence, Sudan Black B, Paraffin sections, Human brain, Fluorescence microscopy