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A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system

Authors

  • Tina Notter,

    1. Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
    2. Neuroscience Center Zurich, Federal Institute of Technology and University of Zurich, Zurich, Switzerland
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    • T.N. and P.P. contributed equally to this study.
  • Patrizia Panzanelli,

    1. Department of Neuroscience Rita Levi Montalcini, University of Turin, Turin, Italy
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    • T.N. and P.P. contributed equally to this study.
  • Sandra Pfister,

    1. Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
    2. Neuroscience Center Zurich, Federal Institute of Technology and University of Zurich, Zurich, Switzerland
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  • Dennis Mircsof,

    1. Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
    2. Neuroscience Center Zurich, Federal Institute of Technology and University of Zurich, Zurich, Switzerland
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  • Jean-Marc Fritschy

    Corresponding author
    1. Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
    2. Neuroscience Center Zurich, Federal Institute of Technology and University of Zurich, Zurich, Switzerland
    • Correspondence: Dr J.-M. Fritschy, 1Institute of Pharmacology and Toxicology, as above.

      E-mail: fritschy@pharma.uzh.ch

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Abstract

Biochemical analysis of central nervous system proteins and nucleic acids requires fresh-tissue homogenates, whereas immunohistochemistry usually is performed in sections prepared from perfusion-fixed tissue. Post-mortem immersion-fixation is possible, but largely impairs morphological preservation and protein antigenicity. Here, we present a simple, fast and versatile protocol allowing concurrent biochemical and immunohistochemical analysis, including pre-embedding immunoelectron microscopy, using tissue from the same animal. The protocol includes a brief transcardiac perfusion with ice-cold, oxygenated and glucose-supplemented artificial cerebrospinal fluid to maintain brain tissue alive, prior to isolation of regions of interest, followed by homogenisation for biochemistry or immersion-fixation for immunohistochemistry. We provide several examples demonstrating that this protocol allows optimal biochemical and morphological analysis, characterised with optimal sensitivity and preservation of tissue structure, along with a reduction of artefacts typically seen in perfusion-fixed tissue. This protocol should find widespread applications for combining analytical methods in tissue from the same animal, thereby reducing the number of mice required for a given experiment.

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