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.

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

PANZANELLI, Patrizia;
2014-01-01

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.
2014
39
2
165
175
Notter T; Panzanelli P; Pfister S; Mircsof D; Fritschy JM.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/142706
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