title: Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections.
abstract: 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.
authors.affiliation: Department of Pathology, Ribeirão Preto School of Medicine - University of São Paulo (USP), Ribeirão Preto - SP, Brazil. vivianecassia@usp.br
authors.forename: V C
authors.initials: VC
authors.lastname: Oliveira
authors.forename: R C V
authors.initials: RC
authors.lastname: Carrara
authors.forename: D L C
authors.initials: DL
authors.lastname: Simoes
authors.forename: F P
authors.initials: FP
authors.lastname: Saggioro
authors.forename: C G
authors.initials: CG
authors.lastname: Carlotti
authors.forename: D T
authors.initials: DT
authors.lastname: Covas
authors.forename: L
authors.initials: L
authors.lastname: Neder
mesh_terms: D000641:Ammonia; D001391:Azo Compounds; D001921:Brain; D004396:Coloring Agents / Q000494:pharmacology*; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D019085:Fluorescent Antibody Technique, Direct; D005557:Formaldehyde; D005629:Frozen Sections; D016591:Histocytological Preparation Techniques / Q000379:methods*; D006652:Histological Techniques; D006801:Humans; D007202:Indicators and Reagents; D009281:Naphthalenes; D009693:Nucleic Acid Hybridization; D016612:Paraffin Embedding; D038761:Photobleaching
chemical_list: D001391:Azo Compounds; D004396:Coloring Agents; D007202:Indicators and Reagents; D009281:Naphthalenes; D005557:Formaldehyde; D000641:Ammonia; C016118:Sudan Black B