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Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics

2009; Elsevier BV; Volume: 8; Issue: 7 Linguagem: Inglês

10.1074/mcp.m800539-mcp200

1535-9484

Jan Mulder, Erik Björling, Kalle Jonasson, Henrik Wernérus, Sophia Hober, Tomas Hökfelt, Mathias Uhlén,

Glycosylation and Glycoproteins Research

A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics. A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics. The brain is the most complex organ in the mammalian body. It processes sensory information from our external environment; produces behavior, emotions, and memories; and regulates the internal body homeostasis. To fulfill these diverse functions the brain harbors a myriad of neuronal networks processing information and connecting input and output systems. Because of the highly specialized functions, each neuron population is neurochemically specified expressing the necessary sets of proteins. Consequently a large number of genes are expressed in the mammalian brain. Based on microarray and in situ hybridization studies it is estimated that ∼55–80% of all mouse genes are expressed in the brain (1Lein E.S. Hawrylycz M.J. Ao N. Ayres M. Bensinger A. Bernard A. Boe A.F. Boguski M.S. Brockway K.S. Byrnes E.J. Chen L. Chen L. Chen T.M. Chin M.C. Chong J. Crook B.E. Czaplinska A. Dang C.N. Datta S. Dee N.R. Desaki A.L. Desta T. Diep E. Dolbeare T.A. Donelan M.J. Dong H.W. Dougherty J.G. Duncan B.J. Ebbert A.J. Eichele G. Estin L.K. Faber C. Facer B.A. Fields R. Fischer S.R. Fliss T.P. Frensley C. Gates S.N. Glattfelder K.J. Halverson K.R. Hart M.R. Hohmann J.G. Howell M.P. Jeung D.P. Johnson R.A. Karr P.T. Kawal R. Kidney J.M. Knapik R.H. Kuan C.L. Lake J.H. Laramee A.R. Larsen K.D. Lau C. Lemon T.A. Liang A.J. Liu Y. Luong L.T. Michaels J. Morgan J.J. Morgan R.J. Mortrud M.T. Mosqueda N.F. Ng L.L. Ng R. Orta G.J. Overly C.C. Pak T.H. Parry S.E. Pathak S.D. Pearson O.C. Puchalski R.B. Riley Z.L. Rockett H.R. Rowland S.A. Royall J.J. Ruiz M.J. Sarno N.R. Schaffnit K. Shapovalova N.V. Sivisay T. Slaughterbeck C.R. Smith S.C. Smith K.A. Smith B.I. Sodt A.J. Stewart N.N. Stumpf K.R. Sunkin S.M. Sutram M. Tam A. Teemer C.D. Thaller C. Thompson C.L. Varnam L.R. Visel A. Whitlock R.M. Wohnoutka P.E. Wolkey C.K. Wong V.Y. Wood M. Yaylaoglu M.B. Young R.C. Youngstrom B.L. Yuan X.F. Zhang B. Zwingman T.A. Jones A.R. Genome-wide atlas of gene expression in the adult mouse brain.Nature. 2007; 445: 168-176Crossref PubMed Scopus (3705) Google Scholar, 2Sandberg R. Yasuda R. Pankratz D.G. Carter T.A. Del Rio J.A. Wodicka L. Mayford M. Lockhart D.J. Barlow C. Regional and strain-specific gene expression mapping in the adult mouse brain.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11038-11043Crossref PubMed Scopus (389) Google Scholar) (gene expression during developmental stages and pathological conditions not included). Interestingly 70% of these genes are expressed in different cell populations each covering less than 20% of the brain, indicating the complexity of the brain and the specialization of individual populations of neurons (1Lein E.S. Hawrylycz M.J. Ao N. Ayres M. Bensinger A. Bernard A. Boe A.F. Boguski M.S. Brockway K.S. Byrnes E.J. Chen L. Chen L. Chen T.M. Chin M.C. Chong J. Crook B.E. Czaplinska A. Dang C.N. Datta S. Dee N.R. Desaki A.L. Desta T. Diep E. Dolbeare T.A. Donelan M.J. Dong H.W. Dougherty J.G. Duncan B.J. Ebbert A.J. Eichele G. Estin L.K. Faber C. Facer B.A. Fields R. Fischer S.R. Fliss T.P. Frensley C. Gates S.N. Glattfelder K.J. Halverson K.R. Hart M.R. Hohmann J.G. Howell M.P. Jeung D.P. Johnson R.A. Karr P.T. Kawal R. Kidney J.M. Knapik R.H. Kuan C.L. Lake J.H. Laramee A.R. Larsen K.D. Lau C. Lemon T.A. Liang A.J. Liu Y. Luong L.T. Michaels J. Morgan J.J. Morgan R.J. Mortrud M.T. Mosqueda N.F. Ng L.L. Ng R. Orta G.J. Overly C.C. Pak T.H. Parry S.E. Pathak S.D. Pearson O.C. Puchalski R.B. Riley Z.L. Rockett H.R. Rowland S.A. Royall J.J. Ruiz M.J. Sarno N.R. Schaffnit K. Shapovalova N.V. Sivisay T. Slaughterbeck C.R. Smith S.C. Smith K.A. Smith B.I. Sodt A.J. Stewart N.N. Stumpf K.R. Sunkin S.M. Sutram M. Tam A. Teemer C.D. Thaller C. Thompson C.L. Varnam L.R. Visel A. Whitlock R.M. Wohnoutka P.E. Wolkey C.K. Wong V.Y. Wood M. Yaylaoglu M.B. Young R.C. Youngstrom B.L. Yuan X.F. Zhang B. Zwingman T.A. Jones A.R. Genome-wide atlas of gene expression in the adult mouse brain.Nature. 2007; 445: 168-176Crossref PubMed Scopus (3705) Google Scholar). The success of humans as a species relies on our mental abilities, a result of brain development during evolution. The human brain is distinguished from other mammalian brains by its size; especially the neocortex involved in higher cognitive functions is greatly enlarged in humans. Despite this difference, the human brain has many similarities to brains of other mammalian species, and to some extent mammalian brains have a well preserved basic architecture (basic uniformity) (for reviews, see Refs. 3Liao B.Y. Zhang J. Evolutionary conservation of expression profiles between human and mouse orthologous genes.Mol. Biol. Evol. 2006; 23: 530-540Crossref PubMed Scopus (165) Google Scholar and 4Premack D. Human and animal cognition: continuity and discontinuity.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13861-13867Crossref PubMed Scopus (211) Google Scholar). Therefore, most human brain nuclei and connections have orthologs in other mammalian species ranging from great apes to rodents. Genetic variation underpins interspecies variation in gene expression and assembly of proteins. The human and rat genomes encode similar numbers of genes of which the majority have persisted throughout evolution without deletion or duplication (5Gibbs R.A. Weinstock G.M. Metzker M.L. Muzny D.M. Sodergren E.J. Scherer S. Scott G. Steffen D. Worley K.C. Burch P.E. Okwuonu G. Hines S. Lewis L. DeRamo C. Delgado O. Dugan-Rocha S. Miner G. Morgan M. Hawes A. Gill R. Celera Holt R.A. Adams M.D. Amanatides P.G. Baden-Tillson H. Barnstead M. Chin S. Evans C.A. Ferriera S. Fosler C. Glodek A. Gu Z. Jennings D. Kraft C.L. Nguyen T. Pfannkoch C.M. Sitter C. Sutton G.G. Venter J.C. Woodage T. Smith D. Lee H.M. Gustafson E. Cahill P. Kana A. Doucette-Stamm L. Weinstock K. Fechtel K. Weiss R.B. Dunn D.M. Green E.D. Blakesley R.W. Bouffard G.G. De Jong P.J. Osoegawa K. Zhu B. Marra M. Schein J. Bosdet I. Fjell C. Jones S. Krzywinski M. Mathewson C. Siddiqui A. Wye N. McPherson J. Zhao S. Fraser C.M. Shetty J. Shatsman S. Geer K. Chen Y. Abramzon S. Nierman W.C. Havlak P.H. Chen R. Durbin K.J. Egan A. Ren Y. Song X.Z. Li B. Liu Y. Qin X. Cawley S. Worley K.C. Cooney A.J. D'Souza L.M. Martin K. Wu J.Q. Gonzalez-Garay M.L. Jackson A.R. Kalafus K.J. McLeod M.P. Milosavljevic A. Virk D. Volkov A. Wheeler D.A. Zhang Z. Bailey J.A. Eichler E.E. Tuzun E. Birney E. Mongin E. Ureta-Vidal A. Woodwark C. Zdobnov E. Bork P. Suyama M. Torrents D. Alexandersson M. Trask B.J. Young J.M. Huang H. Wang H. Xing H. Daniels S. Gietzen D. Schmidt J. Stevens K. Vitt U. Wingrove J. Camara F. Mar Albà M. Abril J.F. Guigo R. Smit A. Dubchak I. Rubin E.M. Couronne O. Poliakov A. Hübner N. Ganten D. Goesele C. Hummel O. Kreitler T. Lee Y.A. Monti J. Schulz H. Zimdahl H. Himmelbauer H. Lehrach H. Jacob H.J. Bromberg S. Gullings-Handley J. Jensen-Seaman M.I. Kwitek A.E. Lazar J. Pasko D. Tonellato P.J. Twigger S. Ponting C.P. Duarte J.M. Rice S. Goodstadt L. Beatson S.A. Emes R.D. Winter E.E. Webber C. Brandt P. Nyakatura G. Adetobi M. Chiaromonte F. Elnitski L. Eswara P. Hardison R.C. Hou M. Kolbe D. Makova K. Miller W. Nekrutenko A. Riemer C. Schwartz S. Taylor J. Yang S. Zhang Y. Lindpaintner K. Andrews T.D. Caccamo M. Clamp M. Clarke L. Curwen V. Durbin R. Eyras E. Searle S.M. Cooper G.M. Batzoglou S. Brudno M. Sidow A. Stone E.A. Venter J.C. Payseur B.A. Bourque G. López-Otín C. Puente X.S. Chakrabarti K. Chatterji S. Dewey C. Pachter L. Bray N. Yap V.B. Caspi A. Tesler G. Pevzner P.A. Haussler D. Roskin K.M. Baertsch R. Clawson H. Furey T.S. Hinrichs A.S. Karolchik D. Kent W.J. Rosenbloom K.R. Trumbower H. Weirauch M. Cooper D.N. Stenson P.D. Ma B. Brent M. Arumugam M. Shteynberg D. Copley R.R. Taylor M.S. Riethman H. Mudunuri U. Peterson J. Guyer M. Felsenfeld A. Old S. Mockrin S. Collins F. Genome sequence of the Brown Norway rat yields insights into mammalian evolution.Nature. 2004; 428: 493-521Crossref PubMed Scopus (1677) Google Scholar). It is evident that small changes in protein structure and altered expression levels of proteins influence brain development and form the basis of interspecies differences. However, most human genes have orthologs in rodents, and for most cell types in the brain their neurochemical specification has been preserved throughout evolution. Because of genomic homology and similarity in basic layout of the mammalian brain as well as the preservation of neurochemical specification of subsets of neurons throughout evolution, animal models have shown their value in medical neurosciences (6Strand A.D. Aragaki A.K. Baquet Z.C. Hodges A. Cunningham P. Holmans P. Jones K.R. Jones L. Kooperberg C. Olson J.M. Conservation of regional gene expression in mouse and human brain.PLoS Genet. 2007; 3: e59Crossref PubMed Scopus (80) Google Scholar). Advances in science are largely dependent on the processing of available information and the generation of new concepts and are driven by innovation and availability of new technologies. Recently mRNA-based techniques have emerged as an effective tool for genome wide analysis of expression levels in entire organs or disease-affected tissue. Results obtained from these studies are a source for identification of novel key molecules and have a predictive value to estimate changes in protein synthesis. There are several ongoing initiatives focusing on the expression profiles of the mammalian brain. The Allen Brain Atlas has produced detailed in situ hybridization profiles for over 20,000 genes in the mouse brain (1Lein E.S. Hawrylycz M.J. Ao N. Ayres M. Bensinger A. Bernard A. Boe A.F. Boguski M.S. Brockway K.S. Byrnes E.J. Chen L. Chen L. Chen T.M. Chin M.C. Chong J. Crook B.E. Czaplinska A. Dang C.N. Datta S. Dee N.R. Desaki A.L. Desta T. Diep E. Dolbeare T.A. Donelan M.J. Dong H.W. Dougherty J.G. Duncan B.J. Ebbert A.J. Eichele G. Estin L.K. Faber C. Facer B.A. Fields R. Fischer S.R. Fliss T.P. Frensley C. Gates S.N. Glattfelder K.J. Halverson K.R. Hart M.R. Hohmann J.G. Howell M.P. Jeung D.P. Johnson R.A. Karr P.T. Kawal R. Kidney J.M. Knapik R.H. Kuan C.L. Lake J.H. Laramee A.R. Larsen K.D. Lau C. Lemon T.A. Liang A.J. Liu Y. Luong L.T. Michaels J. Morgan J.J. Morgan R.J. Mortrud M.T. Mosqueda N.F. Ng L.L. Ng R. Orta G.J. Overly C.C. Pak T.H. Parry S.E. Pathak S.D. Pearson O.C. Puchalski R.B. Riley Z.L. Rockett H.R. Rowland S.A. Royall J.J. Ruiz M.J. Sarno N.R. Schaffnit K. Shapovalova N.V. Sivisay T. Slaughterbeck C.R. Smith S.C. Smith K.A. Smith B.I. Sodt A.J. Stewart N.N. Stumpf K.R. Sunkin S.M. Sutram M. Tam A. Teemer C.D. Thaller C. Thompson C.L. Varnam L.R. Visel A. Whitlock R.M. Wohnoutka P.E. Wolkey C.K. Wong V.Y. Wood M. Yaylaoglu M.B. Young R.C. Youngstrom B.L. Yuan X.F. Zhang B. Zwingman T.A. Jones A.R. Genome-wide atlas of gene expression in the adult mouse brain.Nature. 2007; 445: 168-176Crossref PubMed Scopus (3705) Google Scholar). The Gene Expression Nervous System Atlas (GENSAT) project uses enhanced green fluorescent protein reporter genes incorporated into bacterial artificial chromosome transgenic mice to visualize the expression profiles of the most important genes (7Gong S. Zheng C. Doughty M.L. Losos K. Didkovsky N. Schambra U.B. Nowak N.J. Joyner A. Leblanc G. Hatten M.E. Heintz N. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.Nature. 2003; 425: 917-925Crossref PubMed Scopus (1608) Google Scholar). This strategy can result in the identification of expressing cell types as the detailed morphology of enhanced green fluorescent protein-expressing cells is apparent. The Brain Maps project has a large collection of mammalian and non-mammalian brain maps using “classical” histochemical techniques but also includes a few protein distribution profiles visualized using immunohistochemistry (8Mikula S. Trotts I. Stone J.M. Jones E.G. Internet-enabled high-resolution brain mapping and virtual microscopy.Neuroimage. 2007; 35: 9-15Crossref PubMed Scopus (160) Google Scholar). We previously described the possibilities of using antibodies raised against human proteins on rodent brain tissue (9Mulder J. Wernérus H. Shi T.J. Pontén F. Hober S. Uhlén M. Hökfelt T. Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.Neuroscience. 2007; 146: 1689-1703Crossref PubMed Scopus (11) Google Scholar). Here we show the first efforts to produce detailed proteome wide large scale tissue profiling maps of a mammalian brain using an antibody-based proteomics approach. In addition to the available, mentioned information on mRNA levels (Allen Brain Atlas), gene expression profiles (Gene Expression Nervous System Atlas), and detailed neuroanatomy (Brain Maps), antibody-based proteomics provide new information on cellular and subcellular distribution of gene products. This information will increase general knowledge and understanding of the organization and functioning of the brain. The study is based on antibodies generated as part of the Human Protein Atlas program aimed at exploring the protein expression patterns in normal and cancer tissues using tissue microarray-based immunohistochemistry and fluorescence-based confocal microscopy (10Barbe L. Lundberg E. Oksvold P. Stenius A. Lewin E. Björling E. Asplund A. Pontén F. Brismar H. Uhlén M. Andersson-Svahn H. Toward a confocal subcellular atlas of the human proteome.Mol. Cell. Proteomics. 2008; 7: 499-508Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The Human Proteome Resource center aims to produce monospecific antibodies against every human gene. So far, the distribution profiles of 3,000 proteins in 48 human tissues, including four brain areas (cerebellum, cerebral cortex, the hippocampal formation, and lateral subventricular zone), and 20 cancers are available (Human Protein Atlas). The antibodies generated within the framework of this program are based on antigens selected as unique regions for each individual protein, called protein epitope signature tags (PrESTs) 1The abbreviations used are:PrESTprotein epitope signature tagCNScentral nervous systemDCxdoublecortinGABAγ-aminobutyric acidGADglutamate decarboxylaseGFPgreen fluorescent proteinHiFohippocampal formationPBKPDZ-binding kinasePDZpostsynaptic density-95/discs large/zona occludens-1HPAHuman Protein Atlas. (11Lindskog M. Rockberg J. Uhlén M. Sterky F. Selection of protein epitopes for antibody production.BioTechniques. 2005; 38: 723-727Crossref PubMed Scopus (55) Google Scholar, 12Berglund L. Andrade J. Odeberg J. Uhlén M. The epitope space of the human proteome.Protein Sci. 2008; 17: 606-613Crossref PubMed Scopus (36) Google Scholar). Over 5,000 antibodies have been generated and validated using Western blot analysis and protein arrays (13Björling E. Lindskog C. Oksvold P. Linné J. Kampf C. Hober S. Uhlén M. Pontén F. A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissue.Mol. Cell. Proteomics. 2008; 7: 825-844Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The smaller size of the rat brain allows analysis of many brain areas and exposure of the antibodies to a very wide variety of proteins. Furthermore tissue can be processed under perfect conditions optimizing tissue antigenicity with flawless tissue morphology. protein epitope signature tag central nervous system doublecortin γ-aminobutyric acid glutamate decarboxylase green fluorescent protein hippocampal formation PDZ-binding kinase postsynaptic density-95/discs large/zona occludens-1 Human Protein Atlas. Here we describe the initial large scale mapping of 89 protein distribution profiles in 25 selected rat brain areas. By exposing systematically sampled rat brain tissue to our collection of monospecific antibodies a more detailed protein atlas of the mammalian brain was produced, expanding the four brain areas available in the human protein atlas to 25 brain areas (Fig. 1) involved in higher cognitive functions, sensation, emotion, maintenance of internal homeostasis, sleep, and motor and sexual behaviors. A database portal has been created to show selected images from the various regions of the brain. All experiments on animals conformed to the European Communities Council Directive (86/609/EEC) and were approved by the local ethics committee (Stockholms Norra Djursförsöksetiska Nämnd; N396/06 N397/06). All performed experimental procedures have been described in detail previously (9Mulder J. Wernérus H. Shi T.J. Pontén F. Hober S. Uhlén M. Hökfelt T. Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.Neuroscience. 2007; 146: 1689-1703Crossref PubMed Scopus (11) Google Scholar). Rats were anesthetized by an intraperitoneal injection of a mixture of Hypnorm and midazolam followed by an intracerebroventricular injection of the mitosis inhibitor colchicine (dissolved in 0.9% NaCl solution to a final concentration of 90 µg in 15 µl). Animals were sacrificed 24 h after colchicine injection. Rats were deeply anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg) and were transcardially perfused with 300 ml of fixative composed of 4% paraformaldehyde and 0.2% picric acid in phosphate buffer that was preceded by a short prerinse (50 ml) with calcium-free Tyrode's solution. After dissection, brains were postfixed for 90 min at 4 °C and transferred to 0.01 m phosphate buffer containing 10% sucrose, 0.02% bacitracin, and 0.01% sodium azide for 48 h at 4 °C. After dehydration the brains were quickly frozen using CO2. Coronal sections (14 µm) were cut in a cryomicrotome and thaw mounted on gelatin-alum-coated slides with a sampling interval of 0.7 mm. Specific PrESTs of 100–150 amino acids for each target protein were designed using bioinformatics tools (11Lindskog M. Rockberg J. Uhlén M. Sterky F. Selection of protein epitopes for antibody production.BioTechniques. 2005; 38: 723-727Crossref PubMed Scopus (55) Google Scholar) and available information on the human genome (Ensembl database). PrESTs were carefully selected, transmembrane regions and signal peptides were avoided, and only amino acid sequences with low homology to other human proteins were used to avoid cross-reactivity. Selected PrESTs were recombinantly produced in Escherichia coli and used to immunize rabbits, and monospecific polyclonal antibodies were generated through immunoaffinity purification of the resulting antisera (14Agaton C. Falk R. Höidén Guthenberg I. Göstring L. Uhlén M. Hober S. Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.J. Chromatogr. A. 2004; 1043: 33-40Crossref PubMed Scopus (52) Google Scholar). Detailed immunohistochemistry procedures are described elsewhere (9Mulder J. Wernérus H. Shi T.J. Pontén F. Hober S. Uhlén M. Hökfelt T. Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.Neuroscience. 2007; 146: 1689-1703Crossref PubMed Scopus (11) Google Scholar). Briefly after a quick rinse in PBS, thaw mounted sections were incubated for 16–24 h with the rabbit monospecific antibodies at a standard dilution (1:1,000) followed by 30-min incubation with horseradish peroxidase-conjugated swine anti-rabbit IgG (P0217, Dako, Copenhagen, Denmark). Immunoreactivity was visualized using the tyramide signal amplification system (TSA-Plus, NEL741B001KT, PerkinElmer Life Sciences). All used Human Proteome Resource center antibodies presented in this study are listed in Table I. For double staining immunohistochemistry, single staining (as described above) with antibodies against PDZ-binding kinase (PBK) (1:1,000; HPA005753) and C6orf64 (1:1,000; HPA007959) was followed by an overnight incubation at 4 °C with a goat anti-doublecortin (DCx) (1:200; sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Ki67 (1:1,000; RM-9106, Thermo Scientific, Waltham, MA), guinea pig anti-vasopressin (1:1,000 preadsorbed with 10−5 m oxytocin peptide; 15Elde R. Hökfelt T. Localization of hypophysiotropic peptides and other biologically active peptides within the brain.Annu. Rev. Physiol. 1979; 41: 587-602Crossref PubMed Scopus (96) Google Scholar), and rabbit anti-oxytocin (1:500; 16Sofroniew M.V. Weindl A. Schinko I. Wetzstein R. The distribution of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig brain. I. The classical hypothalamo-neurophypophyseal system.Cell Tissue Res. 1979; 196: 367-384Crossref PubMed Scopus (133) Google Scholar). Immunoreactivity was visualized by incubation with appropriate Rhodamine Red X-conjugated donkey anti-rabbit, anti-guinea pig, or anti-goat antibody (1:200; 711-295-152, Jackson ImmunoResearch Laboratories, West Grove, PA). The combination of tyramide signal amplification with direct fluorescence can be used for double labeling experiments using primary antibodies raised in the same species against proteins expressed in different cells or cellular compartments (17Wang G. Achim C.L. Hamilton R.L. Wiley C.A. Soontornniyomkij V. Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy.Methods. 1999; 18: 459-464Crossref PubMed Scopus (74) Google Scholar). This method was used for double labeling experiments using rabbit antibodies targeted against Ki67 and PBK. All antibodies generated and validated within the Human Protein Atlas project and used in this report will be available for the scientific community and can be purchased via Prestige Antibodies (Sigma-Aldrich) or Atlas Antibodies (Stockholm, Sweden).Table IList of antibodies presented including references to published immunohistochemical data on brain tissueAntibodyGene IDGene description (published)Gene info human/gene info ratPercentWBBand sizeFig.HPA004063AIPAryl hydrocarbon receptor-interacting protein (n/a)ENSG00000110711891+0.6 kDa3TENSRNOG00000022289<1%HPA003229ALS2CR13ALS2 (juvenile) chromosome region, candidate 13 (n/a)ENSG00000138439941+9 kDa3R; 7,A and BENSRNOG0000002206628%HPA000612APOOLApolipoprotein O-like(n/a)ENSG00000155008702+0.4 kDa4BENSRNOG00000004512<1%HPA002317BIRC3Baculoviral IAP repeat-containing 3 (n/a)ENSG00000023445743+0.2 kDa4CENSRNOG00000005731<1%HPA007959C6orf64Chromosome 6 open reading frame 64(n/a)ENSG00000112167901−0.8 kDa6, A–DENSRNOG000000063414%HPA007306CALB2Calbindin 2, 29 kDa (calretinin)(21Résibois A. Rogers J.H. Calretinin in rat brain: an immunohistochemical study.Neuroscience. 1992; 46: 101-134Crossref PubMed Scopus (440) Google Scholar)ENSG000001721371001−3.0 kDa3L; 5, D and EENSRNOG000000169779%HPA007856CCAR1Cell division cycle and apoptosis regulator 1 (n/a)ENSG00000060339941−14.6 kDa3PENSRNOG0000000039711%HPA005551CFHComplement factor H (n/a)ENSG00000000971752−7.2 kDa4AENSRNOG0000003071512%HPA003342CXADRCoxsackie virus and adenovirus receptor (32Hauwel M. Furon E. Gasque P. Molecular and cellular insights into the coxsackie-adenovirus receptor: role in cellular interactions in the stem cell niche.Brain Res. Brain Res. Rev. 2005; 48: 265-272Crossref PubMed Scopus (23) Google Scholar)ENSG000000768648912.5 kDa3BENSRNOG000000015577%HPA001648DDX3XDEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked (n/a)ENSG00000215301971+8.16 kDa3HENSRNOG0000002338311%HPA005835ECH1Enoy-coenzyme A hydratase 1, peroxisomalENSG000001048238117.3 kDa3YENSRNOG0000002030820%HPA002025ERLIN2ER lipid raft-associated 2 (n/a)ENSG0000014747595111.7 kDa3SENSRNOG0000001376331%HPA001911FARSAPhenylalanine-tRNA synthetase-like, α subunit (n/a)ENSG00000179115901+2.7 kDa3FENSRNOG000000031495%HPA000841GABRA3γ-Aminobutyric acid (GABA) A receptor, α3 (n/a)ENSG00000011677931+10.1 kDa3JENSRNOG0000001684523%HPA008001LRPAP1Low density lipoprotein receptor-related protein-associated protein 1 (n/a)ENSG00000163956801+4.6 kDa3WENSRNOG0000000931311%HPA000781LRRC62Leucine-rich repeat-containing 62 (n/a)ENSG00000166897981+11.3 kDa3OENSRNOG0000000793411%HPA005652MSX2Msh homeobox 2 (33Nishikawa K. Nakanishi T. Aoki C. Hattori T. Takahashi K. Taniguchi S. Differential expression of homeobox-containing genes Msx-1 and Msx-2 and homeoprotein Msx-2 expression during chick craniofacial development.Biochem. Mol. Biol. Int. 1994; 32: 763-771PubMed Google Scholar)ENSG00000120149862+10.3 kDa3IENSRNOG0000001835536%HPA002896NDRG2NDRG family member 2 (34Mitchelmore C. Büchmann-Møller S. Rask L. West M.J. Troncoso J.C. Jensen N.A. NDRG2: a novel Alzheimer's disease associated protein.Neurobiol. Dis. 2004; 16: 48-58Crossref PubMed Scopus (110) Google Scholar)ENSG00000165795941+3.4 kDa3MENSRNOG000000103899%HPA001303NMT2N-Myristoyltransferase 2 (n/a)ENSG00000152465821−2.7 kDa3AENSRNOG000000262484%HPA003215OGFOD12-Oxoglutarate and iron-dependent oxygenase domain-containing 1 (n/a)ENSG00000087263812+12.0 kDa3VENSRNOG0000001928819%HPA005753PBKPDZ-binding kinase (20Dougherty J.D. Garcia A.D. Nakano I. Livingstone M. Norris B. Polakiewicz R. Wexler E.M. Sofroniew M.V. Kornblum H.I. Geschwind D.H. PBK/TOPK, a proliferating neural progenitor-specific mitogen-activated protein kinase kinase.J. Neurosci. 2005; 25: 10773-10785Crossref PubMed Scopus (82) Google Scholar)ENSG00000168078941−0.1 kDa5, A–CENSRNOG00000015308<1%HPA001032PDCD4Programmed cell death 4 (neoplastic transformation inhibitor) (n/a)ENSG0000015059398111.1 kDa4DENSRNOG0000001477920%HPA002114PSMD14Proteasome (prosome, macropain) 26 S subunit, non-ATPase, 14 (n/a)ENSG0000011523310010 kDa3CENSRNOG00000004911<1%HPA001922RAP1GAPRAP1 GTPase-activating protein (n/a)ENSG000000768649118.7 kDa3EENSRNOG0000001382511%HPA003372RPL960 S ribosomal protein L9 (n/a)ENSG00000163682981+0.7 kDa3NENSRNOG000000267163%HPA006641SCGNSecretagogin, EF-hand calcium-binding protein (35Gartner W. Lang W. Leutmetzer F. Domanovits H. Waldhäusl W. Wagner L. Cerebral expression and serum detectability of secretagogin, a recently cloned EF-hand Ca(2+)-binding protein.Cereb. Cortex. 2001; 11: 1161-1169Crossref PubMed Scopus (74) Google Scholar)ENSG00000079689961−3.0 kDa3QENSRNOG000000166289%HPA004057SLC6A2Solute carrier family 6 (neurotransmitter transporter, noradrenalin), member 2 (36Lorang D. Amara S.G. Simerly R.B. Cell-type-specific expression of catecholamine transporters in the rat brain.J. Neurosci. 1994; 14: 4903-4914Crossref PubMed Google Scholar)ENSG00000103546933+5.6 kDa3XENSRNOG000000163118%HPA001830SNAP25Synaptosome-associated protein, 25 kDa (37Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billingsley M. Bloom F.E. Wilson M.C. The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (688) Google Scholar)ENSG000001326391001+8 kDa7, C and DENSRNOG0000000603739%HPA001814SOD2Superoxide dismutase 2 (mitochondrial) (38Medina L. Figueredo-Cardenas G. Reiner A. Differential abundance of superoxide dismutase in interneurons versus projection neurons and in matrix versus striosome n