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A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays

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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.

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Acknowledgments

We are indebted to Macarena Solís-Maldonado (Biochemistry Institute, UACh) for technical assistance. We acknowledge Ricardo Silva Riveros and Patricia Valenzuela Rivera (School of Medicine, UACh) for giving us access to electron microscopy sample preparation facilities, and Sergio Mezzano and María Eugenia Burgos-Concha (Medical Institute, General Hospital of Valdivia, UACh) for giving us access to the cryostat. We thank Rubén Gerardo Contreras and John F. Allen for critical reading and suggestions on the manuscript. We also acknowledge the Chilean CONICYT-FONDECYT program for the postdoctoral fellowship #3140218 and DID-UACh S-2015-81 to Abraham Rosas-Arellano and FONDECYT grants #1110571 and #1151206 to Maite A. Castro. Felipe A. Beltrán is a CONICYT fellow. This work was also supported in Mexico by the basic sciences CONACYT project #179835 to Fanis Missirlis.

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Authors and Affiliations

  1. Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile

    Abraham Rosas-Arellano, Juan B. Villalobos-González, Felipe A. Beltrán & Maite A. Castro

  2. Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile

    Abraham Rosas-Arellano, Juan B. Villalobos-González, Felipe A. Beltrán & Maite A. Castro

  3. Unidad de Microscopía, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México

    Lourdes Palma-Tirado & Alfonso Cárabez-Trejo

  4. Departamento de Fisiología, Biofísica y Neurociencias, Cinvestav del IPN, Unidad Zacatenco, Ciudad de México, México

    Fanis Missirlis

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  1. Abraham Rosas-Arellano
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  2. Juan B. Villalobos-González
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  3. Lourdes Palma-Tirado
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  4. Felipe A. Beltrán
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  5. Alfonso Cárabez-Trejo
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  6. Fanis Missirlis
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  7. Maite A. Castro
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Correspondence to Abraham Rosas-Arellano or Maite A. Castro.

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Rosas-Arellano, A., Villalobos-González, J.B., Palma-Tirado, L. et al. A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays. Histochem Cell Biol 146, 421–430 (2016). https://doi.org/10.1007/s00418-016-1447-2

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