A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system
Tina Notter
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
T.N. and P.P. contributed equally to this study.Search for more papers by this authorPatrizia Panzanelli
Department of Neuroscience Rita Levi Montalcini, University of Turin, Turin, Italy
T.N. and P.P. contributed equally to this study.Search for more papers by this authorSandra Pfister
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
Search for more papers by this authorDennis Mircsof
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
Search for more papers by this authorCorresponding Author
Jean-Marc Fritschy
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
Correspondence: Dr J.-M. Fritschy, 1Institute of Pharmacology and Toxicology, as above.
E-mail: [email protected]
Search for more papers by this authorTina Notter
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
T.N. and P.P. contributed equally to this study.Search for more papers by this authorPatrizia Panzanelli
Department of Neuroscience Rita Levi Montalcini, University of Turin, Turin, Italy
T.N. and P.P. contributed equally to this study.Search for more papers by this authorSandra Pfister
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
Search for more papers by this authorDennis Mircsof
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
Search for more papers by this authorCorresponding Author
Jean-Marc Fritschy
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
Correspondence: Dr J.-M. Fritschy, 1Institute of Pharmacology and Toxicology, as above.
E-mail: [email protected]
Search for more papers by this authorAbstract
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.
References
- Baker, H., Grillo, M. & Margolis, F.L. (1989) Biochemical and immunocytochemical characterization of olfactory marker protein in the rodent central nervous system. J. Comp. Neurol., 285, 246–261.
- Bignami, A., Dahl, D. & Rueger, D.C. (1980) Glial fibrillary acidic protein (GFA) in normal neural cells and in pathological conditions. Adv. Cell. Neurobiol., 1, 285–310.
- Celio, M.R. (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience, 35, 375–475.
- Chen, G., Racay, P., Bichet, S., Celio, M.R., Eggli, P. & Schwaller, B. (2006) Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells. Neuroscience, 142, 97–105.
- Chi, P., Greengard, P. & Ryan, T.A. (2001) Synapsin dispersion and reclustering during synaptic activity. Nat. Neurosci., 4, 1187–1193.
- Chung, K. & Deisseroth, K. (2013) CLARITY for mapping the nervous system. Nat. Methods, 10, 508–513.
- Doehner, J., Madhusudan, A., Konietzko, U., Fritschy, J.M. & Knuesel, I. (2010) Co-localization of Reelin and proteolytic APP fragments in hippocampal plaques in aged wild type mice. J. Alzheimers Dis., 19, 1339–1357.
- Dumoulin, A., Rostaing, P., Bedet, C., Lévi, S., Isambert, M.F., Henry, J.P., Triller, A. & Gasnier, B. (1999) Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons. J. Cell Sci., 112, 811–823.
- Duveau, V., Laustela, S., Barth, L., Gianolini, F., Vogt, K.E., Keist, R., Chandra, D., Homanics, G.E., Rudolph, U. & Fritschy, J.M. (2011) Spatiotemporal specificity of GABAA receptor-mediated regulation of adult hippocampal neurogenesis. Eur. J. Neurosci., 34, 362–373.
- Elsaesser, R., Montani, G., Tirindelli, R. & Paysan, J. (2005) Phosphatidyl-inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium. Eur. J. Neurosci., 21, 2692–2700.
- Eyre, M., Renzi, M., Farrant, M. & Nusser, Z. (2012) Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms. J. Neurosci., 32, 5853–5867.
- Fritschy, J.M. & Panzanelli, P. (2006) Molecular and synaptic organization of GABAA receptors in the cerebellum: effects of targeted subunit gene deletions. Cerebellum, 5, 275–285.
- Fritschy, J.M., Weinmann, O., Wenzel, A. & Benke, D. (1998) Synapse-specific localization of NMDA and GABA(A) receptor subunits revealed by antigen-retrieval immunohistochemistry. J. Comp. Neurol., 390, 194–210.
10.1002/(SICI)1096-9861(19980112)390:2<194::AID-CNE3>3.0.CO;2-X CAS PubMed Web of Science® Google Scholar
- Fritschy, J.M., Panzanelli, P., Kralic, J.E., Vogt, K.E. & Sassoè-Pognetto, M. (2006) Differential dependence of axo-dendritic and axo-somatic GABAergic synapses on GABAA receptors containing the α1 subunit in Purkinje cells. J. Neurosci., 26, 3245–3255.
- Hökfelt, T., Johansson, O., Fuxe, K., Goldstein, M. & Park, D. (1977) Immunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain II. Tyrosine hydroxylase in the telencephalon. Med. Biol., 55, 21–40.
- Krstic, D., Madhusudan, A., Doehner, J., Vogel, P., Notter, T., Imhof, C., Manalastas, A., Hilfiker, M., Pfister, S., Schwerdel, C., Riether, C., Meyer, U. & Knuesel, I. (2012a) Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. J. Neuroinflamm., 9, 151.
- Krstic, D., Rodriguez, M. & Knuesel, I. (2012b) Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators. PLoS One, 7, e47793.
- Lidov, H.G., Grzanna, R. & Molliver, M.E. (1980) The serotonin innervation of the cerebral cortex in the rat – an immunohistochemical analysis. Neuroscience, 5, 207–227.
- Lorincz, A. & Nusser, Z. (2008) Specificity of immunoreactions: the importance of testing specificity in each method. J. Neurosci., 28, 9083–9086.
- Nusser, Z., Roberts, J.D., Baude, A., Richards, J.G. & Somogyi, P. (1995) Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method. J. Neurosci., 15, 2948–2960.
- Panzanelli, P., Gunn, B.G., Schlatter, M.C., Benke, D., Tyagarajan, S.K., Scheiffele, P., Belelli, D., Lambert, J.J., Rudolph, U. & Fritschy, J.M. (2011) Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells. J. Physiol., 589, 4959–4980.
- Pfeiffer, F., Simler, R., Grenningloh, G. & Betz, H. (1984) Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proc. Natl. Acad. Sci. USA, 81, 7224–7227.
- Pfister, S., Dietrich, M., Sidler, C., Fritschy, J., Knuesel, I. & Elsaesser, R. (2012) Characterization and turnover of CD73/IP(3)R3-positive microvillar cells in the adult mouse olfactory epithelium. Chem. Senses, 37, 859–868.
- Sassoè-Pognetto, M., Panzanelli, P., Sieghart, W. & Fritschy, J.M. (2000) Co-localization of multiple GABA(A) receptor subtypes with gephyrin at postsynaptic sites. J. Comp. Neurol., 420, 481–498.
10.1002/(SICI)1096-9861(20000515)420:4<481::AID-CNE6>3.0.CO;2-5 CAS PubMed Web of Science® Google Scholar
- Schmittgen, T. & Livak, K. (2008) Analyzing real-time PCR data by the comparative CT method. Nat. Protoc., 8, 1101–1108.
- Schneider Gasser, E.M., Straub, C.J., Panzanelli, P., Weinmann, O., Sassoè-Pognetto, M. & Fritschy, J.M. (2006) Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons. Nat. Protoc., 1, 1887–1897.
- Schneider Gasser, E.M., Duveau, V., Prenosil, G.A. & Fritschy, J.M. (2007) Reorganization of GABAergic circuits maintains GABAA receptor-mediated transmission onto CA1 interneurons in α1-subunit-null mice. Eur. J. Neurosci., 25, 3287–3304.
- Simat, M., Parpan, F. & Fritschy, J.M. (2007) Heterogeneity of glycinergic and GABAergic interneurons in the granule cell layer of mouse cerebellum. J. Comp. Neurol., 500, 71–83.
- Studer, R., von Boehmer, L., Haenggi, T., Schweizer, C., Benke, D., Rudolph, U. & Fritschy, J.M. (2006) Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor α3 subunit-null mice. Eur. J. Neurosci., 24, 1307–1315.
- Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J., Obata, K. & Kaneko, T. (2003) Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol., 467, 60–79.
- Ulvestad, E., Williams, K., Bjerkvig, R., Tiekotter, K., Antel, J. & Matre, R. (1994) Human microglial cells have phenotypic and functional characteristics in common with both macrophages and dendritic antigen-presenting cells. J. Leukocyte Biol., 56, 732–740.
- Varoqueaux, F., Jamain, S. & Brose, N. (2004) Neuroligin 2 is exclusively localized to inhibitory synapses. Eur. J. Cell Biol., 83, 449–456.
- Watanabe, M., Fukaya, M., Sakimura, K., Manabe, T., Mishina, M. & Inoue, Y. (1998) Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield. Eur. J. Neurosci., 10, 478–487.
- Zeilhofer, H.R., Studler, B., Arabadzisz, D., Schweizer, C., Ahmadi, S., Layh, B., Bösl, M.R. & Fritschy, J.M. (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J. Comp. Neurol., 482, 123–141.
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