title: A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system.
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.
authors: Institute of Pharmacology and Toxicology, University of Zurich, CH-8057, Zurich, Switzerland; Neuroscience Center Zurich, Federal Institute of Technology and University of Zurich, Zurich, Switzerland.
authors: Tina
authors: T
authors: Notter
authors: Patrizia
authors: P
authors: Panzanelli
authors: Sandra
authors: S
authors: Pfister
authors: Dennis
authors: D
authors: Mircsof
authors: Jean-Marc
authors: JM
authors: Fritschy
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure*; D001923:Brain Chemistry; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis; D016326:Extracellular Matrix Proteins / Q000032:analysis; D020869:Gene Expression Profiling / Q000379:methods; D005968:Glutamate Decarboxylase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016253:Microscopy, Immunoelectron / Q000379:methods; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000737:chemistry / Q000166:cytology; D010477:Perfusion; D060888:Real-Time Polymerase Chain Reaction; D011963:Receptors, GABA-A / Q000032:analysis; D000091042:Reelin Protein; D012697:Serine Endopeptidases / Q000032:analysis; D013347:Subcellular Fractions / Q000737:chemistry; D014021:Tissue Preservation
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D011963:Receptors, GABA-A; D000091042:Reelin Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1
keywords: RT-PCR; Western blot; antibodies; immunofluorescence; ultrastructure