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A protocol for concurrent high‐quality immunohistochemical and biochemical analyses in adult mouse central nervous system
Author(s) -
Notter Tina,
Panzanelli Patrizia,
Pfister Sandra,
Mircsof Dennis,
Fritschy JeanMarc
Publication year - 2014
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/ejn.12447
Subject(s) - immunohistochemistry , biology , pathology , fixative , fixation (population genetics) , brain tissue , central nervous system , immunoelectron microscopy , perfusion , anatomy , medicine , biochemistry , neuroscience , staining , gene
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.