DIG_InSitu_ManualCover_RZ 30.07.2008 17:28 Uhr Seite 3 C M Y CM MY CY CMY K DIG Application Manual for Nonradioactive In Situ Hybridization 4th Edition Probedruck DIG_InSitu_ManualCover_RZ 30.07.2008 17:28 Uhr Seite 4 C M Y CM MY CY CMY K Intended use Our preparations are exclusively intended to be used in life science research applications.* They must not be used in or on human beings since they were neither tested nor intended for such utilization. Preparations with hazardous substances Our preparations may represent hazardous substances to work with. The dangers which, to our knowledge, are involved in the handling of these preparations (e.g., harmful, irritant, toxic, etc.), are separately mentioned on the labels of the packages or on the pack inserts; if for certain preparations such danger references are missing, this should not lead to the conclusion that the corresponding preparation is harmless. All preparations should only be handled by trained personnel. Preparations of human origin The material has been prepared exclusively from blood that was tested for Hbs antigen and for the presence of antibodies to the HIV-1, HIV-2, HCV and found to be negative. Nevertheless, since no testing method can offer complete assurance regarding the absence of infectious agents, products of human origin should be handled in a manner as recommended for any potentially infectious human serum or blood specimen. Liability The user is responsible for the correct handling of the products and must follow the instructions of the pack insert and warnings on the label. Roche Diagnostics shall not assume any liability for damages resulting from wrong handling of such products. Because of its outstanding tactile sensitivity, the Red-Eyed-Tree Frog (Agalychnis callidryas) was chosen to represent the high sensitivity of the DIG System. * exeption: instruments specifically intended for in-vitro diagnostic use. Impressum © 2008 by Roche Diagnostics GmbH Editorial Management: Doris Eisel Oliver Seth, PH.D. Stefanie Grünewald-Janho Bettina Kruchen, PH.D. Art Direction and Design: Designgruppe Fanz & Neumayer Schifferstadt Layout and Typesetting: ACTIVE ARTWARE Gruppe Saarbrücken Probedruck DIG Application Manual for Nonradioactive In Situ Hybridization 4th Edition __DIGDIG AApplicationpplication MManualanual 33rd.indbrd.indb 1 229.07.20089.07.2008 117:37:557:37:55 __DIGDIG AApplicationpplication MManualanual 33rd.indbrd.indb 2 229.07.20089.07.2008 117:38:007:38:00 Table of Contents Chapter 1 General Introduction to General Introduction to In Situ Hybridization .................................................................8 In Situ Hybridization Introduction to Hapten Labeling and Detection of Nucleic Acids ........................................................................................10 Choosing the Right Labeling Method for your Hybridization Experiment....................................................................................13 Chapter 2 Guidelines for In Situ Guidelines for In Situ Hybridization .................................................................................18 Hybridization Details of the Technique ......................................................................................................18 Chapter 3 Nucleic Acid Hybridization – Nucleic Acid Hybridization – General Aspects ...........................................................28 General Aspects Chapter 4 Procedures for Labeling DNA, I. Random primed labeling of ds DNA with DIG-, RNA, and Oligonucleotides with Biotin- or Fluorescein-High Prime reaction mix ...............................................36 DIG, Biotin, or Fluorochromes II. PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes ....................................................................................................38 III. Nick-translation labeling of ds DNA with Nick Translation Mixes for in situ Probes ............................................................45 IV. Nick-translation labeling of ds DNA with DIG-, Biotin-, or Fluorochrome-labeled dUTP ...............................................................47 V. RNA labeling by in vitro transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix .........................................49 VI. Oligonucleotide 3’-end labeling with DIG-ddUTP or Biotin-ddUTP ....................................................................................53 VII. Oligonucleotide tailing with a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP mixture .........................................................56 VIII. Estimating the yield of DIG-labeled nucleic acids ...........................................59 IX. Purifi cation of labeled probes using the High Pure PCR Product Purifi cation Kit ...............................................................65 3 __DIGDIG AApplicationpplication MManualanual 33rd.indbrd.indb 3 229.07.20089.07.2008 117:38:007:38:00 Table of Content Chapter 5 Procedures for In Situ Procedures for In Situ Hybridization to Chromosomes, Hybridization to Chromosomes, Cells, and Tissue Sections ...................................................................................................68 Cells, and Tissue Sections ISH to whole chromosomes In situ hybridization to human metaphase chromo somes using DIG-, biotin-, or fl uorochrome-labeled DNA probes and detection with fl uoro chrome conjugates .............................................................69 J. Wiegant, Department of Cytochemistry and Cytometry, Leiden University, Netherlands. Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension .........................................................................85 D. Celeda1, 2, U. Bettag1, and C. Cremer1 1 Institute for Applied Physics, University of Heidelberg. 2 Institute for Human Genetics and Anthropology, University of Heidelberg, Germany. A simplifi ed and effi cient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe ......................................................................................................89 Prof. Dr. E. R. Schmidt, Institute for Genetics, Johannes Gutenberg-University of Mainz, Germany. Multiple-target DNA in situ hybridization with enzyme-based cyto chemical detection systems .......................................................................................94 E. J. M. Speel, F. C. S. Ramaekers, and A. H. N. Hopman, Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands DNA in situ hybridization with an alkaline phosphatase-based fl uorescent detection system ..........................................................................................105 Dr. G. Sagner, Research Laboratories, Roche GmbH, Penzberg, Germany. ISH to cells Combined DNA in situ hybridization and immuno cytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations.............................................................. 108 E. J. M. Speel, F. C. S. Ramaekers, and A. H. N. Hopman, Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands In situ hybridization to mRNA in in vitro cultured cells with DNA probes ................................................................................................................ 116 Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands. Identifi cation of single bacterial cells using DIG-labeled oligo nucleotides ......................................................................................... 119 B. Zarda, Dr. R. Amann, and Prof. Dr. K.-H. Schleifer, Institute for Microbiology, Technical University of Munich, Germany. 4 DIG Application Manual for In Situ Hybridization _DIG Application Manual 3rd.indb 4 29.07.2008 17:38:00 Table of Content ISH to tissues Detection of HPV 11 DNA in paraffi n-embedded laryngeal tissue with a DIG-labeled DNA probe .....................................................................................123 Dr. J. Rolighed and Dr. H. Lindeberg, ENT-department and Institute for Pathology, Aarhus University Hospital, Denmark. Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes............................................................................................. 129 P. Komminoth, Division of Cell and Molecular Pathology, Department of Pathology, University of Zürich, Switzerland. Detection of mRNA on paraffi n embedded material of the central nervous system with DIG-labeled RNA probes ................................144 H. Breitschopf and G. Suchanek, Research Unit for Experimental Neuropathology, Austrian Academy of Sciences, Vienna, Austria. RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction ..................... 151 Marc DeBlock and Dirk Debrouwer, Plant Genetic Systems N.V., Gent, Belgium. Detection of neuropeptide mRNAs in tissue sections using oligo-nucleotides tailed with fl uorescein-12-dUTP or DIG-dUTP ....................157 Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands. RNA in situ hybridization using DIG-labeled cRNA probes ...............................160 H. B. P. M. Dijkman, S. Mentzel, A. S. de Jong, and K. J. M. Assmann Department of Pathology, Nijmegen University Hospital, Nijmegen, The Netherlands.