ABSTRACT
Hybridization of paraf n-or plastic-embedded tissue sections with labeled probes to visualize the cellular distribution of mRNA transcripts is known as in situ hybridization (ISH). The following ISH protocol is based on that of Jackson,1 but it includes a number of modi cations to improve results from woody plant tissues. Special features of this protocol include the use of full-length RNA probes at high hybridization temperatures, followed, when colorimetric detection is used, by color development in buffer containing polyvinyl alcohol (PVA) to reduce chromagen diffusion.2 These modi cations reduce background from nonspeci c hybridization and reduce color development time while improving signal localization. The protocol works well with soft tissues and eliminates background due to nonspeci c probe sticking in secondary vascular tissues of woody plant specimens. The method is applicable to animal tissues as well. RNase-free technique (see Chapter 8)
14.1 Introduction .......................................................................................................................... 273 14.2 Tissue Preparation ................................................................................................................ 274
14.2.1 Tissue Collection and Fixation ................................................................................. 274 14.2.2 Dehydration Using an Ethanol/Tert-Butanol/H2O Series ......................................... 274
14.3 In ltration, Embedding, and Section Preparation ................................................................ 275 14.3.1 Paraf n In ltration and Embedding ......................................................................... 275 14.3.2 Plastic In ltration and Embedding ........................................................................... 275 14.3.3 Sectioning ................................................................................................................. 275
14.4 Hybridization ........................................................................................................................ 276 14.4.1 Paraf n or Plastic Removal and Section Rehydration .............................................. 276 14.4.2 Heat Denaturation and Protease Treatment .............................................................. 276 14.4.3 Acetylation for Nonspeci c Blocking....................................................................... 277 14.4.4 RNA Probe Preparation ............................................................................................ 277 14.4.5 Hybridization ............................................................................................................ 279
14.5 Nonradioactive Signal Detection ..........................................................................................280 14.5.1 Post-Hybridization Washing and Blocking ...............................................................280 14.5.2 Immunodetection of DIG Probes ..............................................................................280 14.5.3 Post-Antibody Washing and Color Development ..................................................... 281
14.6 Radioactive Signal Detection ............................................................................................... 281 14.6.1 Post-Hybridization Washing ..................................................................................... 281 14.6.2 Autoradiography ....................................................................................................... 282 14.6.3 Staining and Viewing ............................................................................................... 283
14.7 Troubleshooting Guide .........................................................................................................285 14.8 Reagents Needed ..................................................................................................................285 References ......................................................................................................................................288
