A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system
| Autor: | Patrizia Panzanelli, Sandra Pfister, Jean-Marc Fritschy, Dennis Mircsof, Tina Notter |
|---|---|
| Přispěvatelé: | University of Zurich |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Pathology
medicine.medical_specialty Cell Adhesion Molecules Neuronal Immunoelectron microscopy Blotting Western Green Fluorescent Proteins Central nervous system 10050 Institute of Pharmacology and Toxicology Mice Transgenic Nerve Tissue Proteins 610 Medicine & health Biology Real-Time Polymerase Chain Reaction Immunofluorescence Mice 03 medical and health sciences 0302 clinical medicine Western blot medicine Animals Microscopy Immunoelectron 030304 developmental biology Brain Chemistry Neurons Extracellular Matrix Proteins 0303 health sciences medicine.diagnostic_test Glutamate Decarboxylase Gene Expression Profiling General Neuroscience Serine Endopeptidases Brain 2800 General Neuroscience Receptors GABA-A Immunohistochemistry Mice Inbred C57BL Perfusion Reelin Protein Real-time polymerase chain reaction medicine.anatomical_structure Ultrastructure 570 Life sciences biology Tissue Preservation 030217 neurology & neurosurgery Subcellular Fractions |
| Zdroj: | The European journal of neuroscience |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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