Commentary Simple and Fast In Situ Hybridization JÚLIO BORLIDO, SUSANA PEREIRA * , ROSÁRIO FERREIRA, NATÁLIA COELHO, PATRÍCIA DUARTE and JOSÉ PISSARRA Institute for Molecular and Cell Biology (IBMC) and Department of Botany, Faculty of Sciences, University of Porto Abstract. RNA in situ hybridization is a powerful technique but can be time consuming and potentially hazardous. We developed a procedure in which nonisotopic in situ hybrid- ization is performed in Vibratome-sectioned tissues that are not dehydrated or embedded in paraffin or resin, thus avoiding additional pretreatment steps to allow the probes to pen- etrate the cell structure. Predigestion with proteases was not necessary. Hybridization of sections and immunologic detection were performed in microplates to avoid the use of slide-coating agents. Biotinylated cDNA probes were synthesized by PCR. Synthetic oligonucleotide probes chemically labeled with biotin or digoxigenin were also used. Stained sections were mounted on slides for microscopic observation, and various tran- scripts were successfully detected using different visualization methods. This method is fast and can be easily implemented in other laboratories because it requires minimal exper- tise in processing biological samples for microscopy. Key words: biotin, cardosin A, digoxigenin, glutamine synthetase, mRNA localization, nonisotopic in situ hybridization, oligonucleotide probes, transgene expression Abbreviations: AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; DIG, digoxigenin; GS, glutamine synthetase; NBT, nitroblue tetrazolium chloride; PBS, phosphate buffer saline; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); UTR, untrans- lated region. In situ hybridization Borlido et al. Introduction In situ hybridization enables the detection of specific nucleic acid sequences in morphologically preserved biological material, providing their exact locations. In situ methods that allow the visualization of the spatial distribution of RNA ex- pression make it possible to evaluate differential gene expression in tissues, cells, and subcellular compartments and to correlate this information with temporal and spatial cell differentiation and specificity. mRNA detection is difficult because it is not spatially concentrated in the cell (eg, chromosomal DNA). When designing in situ hybridization experiments, researchers must consider items such as tissue fixation and embedding, section- ing, probe labeling, endogenous biotin or enzyme activity, and desired sensitivity. Plant Molecular Biology Reporter 20: 219–229, September 2002 © 2002 International Society for Plant Molecular Biology. Printed in Canada. * Author for correspondence. Present address: IBMC, Rua Campo Alegre, 823, 4150-180 Porto, Portugal; e-mail: spereira@ibmc.up.pt; fax: 351-226099157; ph: 351-226074900.