title: A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays.
abstract: Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.
authors.affiliation: Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. rosas.arellano@gmail.com.|Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile. rosas.arellano@gmail.com.
authors.forename: Abraham
authors.identifier: http://orcid.org/0000-0003-1422-9538
authors.initials: A
authors.lastname: Rosas-Arellano
authors.affiliation: Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.|Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile.
authors.forename: Juan B
authors.initials: JB
authors.lastname: Villalobos-González
authors.affiliation: Unidad de Microscopía, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Palma-Tirado
authors.affiliation: Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.|Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile.
authors.forename: Felipe A
authors.initials: FA
authors.lastname: Beltrán
authors.affiliation: Unidad de Microscopía, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Cárabez-Trejo
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Cinvestav del IPN, Unidad Zacatenco, Ciudad de México, México.
authors.forename: Fanis
authors.initials: F
authors.lastname: Missirlis
authors.affiliation: Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. macastro@uach.cl.|Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile. macastro@uach.cl.
authors.forename: Maite A
authors.initials: MA
authors.lastname: Castro
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D005455:Fluorescent Antibody Technique / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron / Q000379:methods*
chemical_list: D000906:Antibodies
keywords: Background staining; Confocal; Fluorescence; Immunohistochemistry; Signal-to-noise ratio; Transmission electron microscopy