Name: in situ hybridization for the precise localization of transcripts in plants
Uploaded: 2011-11-23T13:00:00
Duration: 12 min 15 s
Description: Watch this Scientific Journal Video about In Situ Hybridization for the Precise Localization of Transcripts in Plants at JoVE.com

Research in the laboratory of M.T. is supported by grants from the National Science Foundation (DBI-0820610 and IOS-1022102) and the NY Department of Health (NYSTEM-C024308). C.M. received postdoctoral fellowships from the Spanish Ministry of Education and Science (2007-0937) and the Foundation Rafael del Pino, Spain.

<ol>
	<li>Rensink, W. A., Buell, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+expression+profiling+resources+for+plant+genomics.">Microarray expression profiling resources for plant genomics.</a> Trends. Plant. Sci. 10, 603-609 (2005).</li><li>Li, P., Ponnala, L., Gandotra, N., Wang, L., Si, Y., Tausta, S. L., Kebrom, T. H., Provart, N., Patel, R., Myers, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+dynamics+of+the+maize+leaf+transcriptome.">The developmental dynamics of the maize leaf transcriptome.</a> Nat. Genet. 42, 1060-1067 (2010).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+integrated+single-base+resolution+maps+of+the+epigenome+in+Arabidopsis.">Highly integrated single-base resolution maps of the epigenome in Arabidopsis.</a> Cell. 133, 523-536 (2008).</li><li>Brady, S. M., Orlando, D. A., Lee, J. Y., Wang, J. Y., Koch, J., Dinneny, J. R., Mace, D., Ohler, U., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+root+spatiotemporal+map+reveals+dominant+expression+patterns.">A high-resolution root spatiotemporal map reveals dominant expression patterns.</a> Science. 318, 801-806 (2007).</li><li>Brooks, L., Strable, J., Zhang, X., Ohtsu, K., Zhou, R., Sarkar, A., Hargreaves, S., Elshire, R. J., Eudy, D., Pawlowska, T., Ware, D., Janick-Buckner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+of+shoot+meristem+functional+domains.">Microdissection of shoot meristem functional domains.</a> PLoS. Genet. 5, e1000476-e1000476 (2009).</li><li>Galbraith, D. W., Birnbaum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+studies+of+cell+type-specific+gene+expression+in+plants.">Global studies of cell type-specific gene expression in plants.</a> Annu. Rev. Plant. Biol. 57, 451-475 (2006).</li><li>Ohtsu, K., Smith, M., Emrich, S., Borsuk, L., Zhou, R., Chen, T., Zhang, X., Timmermans, M., Beck, J., Buckner, B., Janick-Buckner, D., Nettleton, D., Scanlon, M., Schnable, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+of+the+shoot+apical+meristem+of+maize+(Zea+mays+L.).">Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.).</a> Plant. J. 52, 391-404 (2007).</li><li>Juarez, M. T., Kui, J. S., Thomas, J., Heller, B. A., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-mediated+repression+of+rolled+leaf1+specifies+maize+leaf+polarity.">microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity.</a> Nature. 428, 84-88 (2004).</li><li>Kramer, E. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+genetic+mechanisms+controlling+petal+development.">Evolution of genetic mechanisms controlling petal development.</a> Nature. 399, 144-148 (1999).</li><li>Stuurman, J., Jäggi, F., Kuhlemeier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoot+meristem+maintenance+is+controlled+by+a+GRAS-gene+mediated+signal+from+differentiating+cells.">Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells.</a> Genes Dev. 16, 2213-2218 (2002).</li><li>Kim, M., McCormick, S., Timmermans, M., Sinha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+domain+of+PHANTASTICA+determines+leaflet+placement+in+compound+leaves.">The expression domain of PHANTASTICA determines leaflet placement in compound leaves.</a> Nature. 424, 438-443 (2003).</li><li>Kramer, E. M., Holappa, L., Gould, B., Jaramillo, M. A., Setnikov, D., Santiago, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elaboration+of+B+gene+function+to+include+the+identity+of+novel+floral+organs+in+the+lower+eudicot+Aquilegia.">Elaboration of B gene function to include the identity of novel floral organs in the lower eudicot Aquilegia.</a> Plant Cell. 19, 750-766 (2007).</li><li>Lippman, Z. B., Cohen, O., Alvarez, J. P., Abu-Abied, M., Pekker, I., Paran, I., Eshed, Y., Zamir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+a+compound+inflorescence+in+tomato+and+related+nightshades.">The making of a compound inflorescence in tomato and related nightshades.</a> PLoS Biol. 6, e288-e288 (2008).</li><li>Kidner, C., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+as+a+tool+to+study+the+role+of+microRNAs+in+plant+development.">In situ hybridization as a tool to study the role of microRNAs in plant development.</a> Methods. Mol. Biol. 342, 159-179 (2006).</li><li>Jackson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+labeling+of+KNOTTED1+mRNA+and+protein+reveals+multiple+potential+sites+of+protein+trafficking+in+the+shoot.">Double labeling of KNOTTED1 mRNA and protein reveals multiple potential sites of protein trafficking in the shoot.</a> 129, 1423-1429 (2002).</li><li>Jensen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Botanical Histochemistry. , 408-408 (1962).</li><li>Jackson, D., Bowles, D. J., Gurr, S. J., McPherson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+in+plants.">In situ hybridization in plants.</a> Molecular Plant Pathology: A Practical Approach. , 163-174 (1991).</li><li>Chitwood, D. H., Nogueira, F. T., Howell, M. o. n. t. g. o. m. e. r. y., Carrington, T. A., C, J., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+via+small+RNA+mobility.">Pattern formation via small RNA mobility.</a> Genes. Dev. 23, 549-554 (2009).</li><li>Piette, D., Hendrickx, M., Willems, E., Kemp, C. R., Leyns, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+procedure+for+whole-mount+in+situ+hybridization+on+mouse+embryos+and+embryoid+bodies.">An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies.</a> Nat. Protoc. 3, 1194-1201 (2008).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Jackson, D., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+phyllotaxy+in+maize+by+the+abphyl1+gene.">Control of phyllotaxy in maize by the abphyl1 gene.</a> Development. , 126-315 (1999).</li><li>Chuck, G., Whipple, C., Jackson, D., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maize+SBP-box+transcription+factor+encoded+by+tasselsheath4+regulates+bract+development+and+the+establishment+of+meristem+boundaries.">The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries.</a> Development. 137, 1243-1250 (2010).</li><li>Long, J. A., Barton, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+apical+embryonic+pattern+in+Arabidopsis.">The development of apical embryonic pattern in Arabidopsis.</a> Development. 125, 3027-3035 (1998).</li><li>Nogueira, F. T., Chitwood, D. H., Madi, S., Ohtsu, K., Schnable, P. S., Scanlon, M. J., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+small+RNA+accumulation+in+the+maize+shoot+apex.">Regulation of small RNA accumulation in the maize shoot apex.</a> PLoS Genet. 5, e1000320-e1000320 (2009).</li></ol>

No conflicts of interest declared.

The in situ hybridization method outlined here allows the direct visualization of the spatiotemporal expression pattern of any gene of interest with great cellular resolution, high sensitivity and specificity. The protocol does not allow a quantitative comparison of expression levels between genes. However, the method's high sensitivity and resolution can reveal gradients of gene expression within a tissue8, 18, which is not possible with most other methods of analyzing gene expression. 
The protocol includes many steps, which can make trouble-shooting problematic. The most critical steps of the protocol are the fixation of the tissue and the selection and labeling of the probe. Poorly infiltrated tissues and probes with low DIG incorporation will yield very weak signals that might be difficult to detect above background. For each new gene of interest, it is advisable to try a few distinct probes derived from different regions of the transcript. Occasionally, two or three probes targeting different smaller regions of the gene may need to be combined to obtain a strong and specific hybridization signal. In addition, the hybridization conditions, such as the temperature and composition of the hybridization buffer, can be optimized for each gene or tissue to lower or increase the hybridization stringency. Also the protease treatment step is a variable and can be changed to adapt the protocol for different plant species or for the detection of transcripts with very low abundance. The inclusion of both positive and negative control probes will be helpful in identifying problematic points of the protocol. 
The procedure is optimized for paraffin-embedded plant tissue sections but with minor modifications can be used also for whole-mount in situ hybridization. Although widely used in animal research19, whole mount in situ hybridization will be limited to plant tissues that can be easily infiltrated, such as embryos, roots or young meristematic tissues20,21. The protocol can also be easily modified to simultaneously detect two distinct mRNAs or localize transcripts alongside proteins15,22,23. Finally, it is possible to extend the application of this method to the visualization of small RNA expression patterns in plants. miRNA expression patterns have been determined using this protocol in both maize and Arabidopsis8,14. More recently, the in vitro transcribed probes have been replaced by commercially available DIG-labeled locked nucleic acid (LNA) oligo probes that increase signal sensitivity18, 24.

<table border="1">
<tbody>
<tr>
<th>Product</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Remarks</th>
</tr>
<tr>
<td>Cytoseal 60 4oz</td>
<td>Fisher</td>
<td>23-244-256</td>
<td>Toluene-based 8310-4</td>
</tr>
<tr>
<td>Histoclear		</td>
<td>Fisher</td>
<td>50-899-90147</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tissue Path Paraplast Plus	 	</td>
<td>Fisher</td>
<td>23-021-400</td>
<td>&nbsp;</td>
</tr>
<tr>
<td> N+ nylon membrane Hybond-N+		 </td>
<td>GE Healthcare</td>
<td>RPN203B	</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>RNase OUT; 40 U/&mu;L			 	</td>
<td>Invitrogen</td>
<td>10777019</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>RQ1 RNase-Free DNase; 1 U/&mu;l 			 	</td>
<td>Promega</td>
<td>M6101</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DIG-labeled Control RNA 			</td>
<td>Roche</td>
<td>11585746910 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DIG RNA labeling mix</td>
<td>Roche</td>
<td>11277073910</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>SP6 RNA polymerase 1,000 U		 	</td>
<td>Roche</td>
<td>10810274001	</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>T3 RNA polymerase 1,000 U		 	</td>
<td>Roche</td>
<td>11031163001	</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>T7 RNA polymerase 1,000 U		 	</td>
<td>Roche</td>
<td>10881767001	</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>RNase-A 				 	</td>
<td>Roche</td>
<td>109142</td>
<td>dissolve in glycerol 50% Tris 100mM pH8.0</td>
</tr>
<tr>
<td>Blocking Reagent 			</td>
<td>Roche</td>
<td>1 096 176</td>
<td>Heat up to 70&deg;C in TBS buffer</td>
</tr>
<tr>
<td>Anti-Digoxygenin-AP, Fab fragments from sheep 150 U		 	</td>
<td>Roche</td>
<td>1 093 274</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NBT/BCIP stock solution			 	</td>
<td>Roche</td>
<td>1 681 451</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>mini Quick Spin RNA Columns 	 	</td>
<td>Roche</td>
<td>11814427001	</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>tRNA from E. coli 	 		 	</td>
<td>Roche</td>
<td>109541</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Triton&reg;X-100 			 	</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tween-20 	</td>
<td>Sigma</td>
<td>P9416</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Deionized formamide			 	</td>
<td>Sigma</td>
<td>F9037</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Protease from Streptomyces griseus Type XIV	 	</td>
<td>Sigma</td>
<td>P5147</td>
<td>Dilute in milliQ water and pre-digest 4 hours at 37&deg;C </td>
</tr>
<tr>
<td>Triethanolamine 	 	</td>
<td>Sigma</td>
<td>90279</td>
<td>Dilute in water, bring to pH8.0 with HCl</td>
</tr>
<tr>
<td>Acetic anhydride 	 	</td>
<td>Sigma		 </td>
<td>A6404</td>
<td>&nbsp;</td>
</tr>
<tr>
<td> Dextran sulfate sodium salt from Leuconostoc spp.			 	</td>
<td>Sigma</td>
<td>D8906-5G</td>
<td>Dextran Sulfate should be heated to 80&deg;C to dissolve.</td>
</tr>
<tr>
<td>Denhardt&#x2019;s Solution 50x Concentrate			 	</td>
<td>Sigma</td>
<td>D2532</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Albumin from bovine serum - &ge;98%			 	</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Eosin Y disodium salt				</td>
<td>Sigma</td>
<td>E4382</td>
<td>Dissolve in 96-100% ethanol until saturated	 </td>
</tr>
<tr>
<td>Paraformaldehyde 	 	</td>
<td>	Sigma</td>
<td>	P6148</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ethanol absolute 200 proof	 	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Phenol/chloroform	 	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Base moulds			 	</td>
<td>Electronic Microsocpy Science</td>
<td>62352-15</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Embedding rings			 	</td>
<td>Fisher</td>
<td>22-038-197</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>20ml disposable scintillation vials, with caps			 	</td>
<td>VWR</td>
<td>66020-326</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Probe-on-plus slides			 	</td>
<td>Fisher</td>
<td>22-230-900</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Micro cover glasses 24x50 mm		 	</td>
<td>VWR</td>
<td>48404-452</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Whatman paper 3mm		 	</td>
<td>VWR</td>
<td>89022-360</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Three glass dishes and one stainless steel slide rack	 	</td>
<td>Electronic Microsocpy Science</td>
<td>71420-25</td>
<td>Two glass dished are necessary for histoclear treatment and the last one is used for the acetic anhydride treatment</td>
</tr>
<tr>
<td>18 clean plastic containers			 	</td>
<td>Electronic Microsocpy Science</td>
<td>71402</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2 or 3 large flat plastic boxes with sealable lids		 	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>required for the hybridization and antibody steps</td>
</tr>

<tr>
<td>Fine brushes 	 	 			 	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Help handing wax ribbons</td>
</tr>

<tr>
<td>Microtome			 	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Leica</td>
</tr>

<tr>
<td>Self-cleaning dry vacuum system				 	</td>
<td>Welch</td>
<td>2025</td>
<td>&nbsp;</td>
</tr>

<tr>
<td>Slide warmer		 		 	</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>

<tr>
<td>Heating block 	 	 			 	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>needed at 60&deg;C and 85&deg;C</td>
</tr>

<tr>
<td>Incubator at 58-60&deg;C		 			 	</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>For steps with molten Paraplast</td>

</tr>

<tr>
<td>Ovens or water baths for 55&deg;C and 37&deg;C 		 			 	</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>Need two due to the quick change between temperatures</td>
</tr>
<tr>
<td>Centrifuge (4&deg;C - 20&deg;C)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fume hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>


</tbody>
</table>

in situ hybridization for the precise localization of transcripts in plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

Watch this Scientific Journal Video about In Situ Hybridization for the Precise Localization of Transcripts in Plants at JoVE.com

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...