Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections.
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.
Oliveira
V C
VC
Department of Pathology, Ribeirão Preto School of Medicine - University of São Paulo (USP), Ribeirão Preto - SP, Brazil. vivianecassia@usp.br
Carrara
R C V
RC
Simoes
D L C
DL
Saggioro
F P
FP
Carlotti
C G
CG
Covas
D T
DT
Neder
L
L
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
D001391:Azo Compounds; D004396:Coloring Agents; D007202:Indicators and Reagents; D009281:Naphthalenes; D005557:Formaldehyde; D000641:Ammonia; C016118:Sudan Black B