We are very grateful to Anders Bovin who provided the music for our film and to Michael Elliot Stocks for volunteering to be the speaker voice. Support was provided by Carl Trygger Foundation, the strategic research program Agricultural Functional Genomics (AgriFunGen) at the Swedish University of Agricultural Sciences, the Swedish Research Council (VR), and the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS).

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J. <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 Patology: a practical approach. , (1991).</li><li>Leitch, A. R., Schwarzacher, T., Jackson, D., Leitch, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization: a practical guide. , (1994).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> Plant Cell. 2 (8), 755-767 (1990).</li><li>Azevedo, H., Lino-Neto, T., Tavares, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+high-quality+RNA+isolation+from+needles+of+adult+maritime+pine+trees.">An improved method for high-quality RNA isolation from needles of adult maritime pine trees.</a> Plant Molecular Biology Reporter. , 333-338 (2003).</li><li>Sundstrom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+active+in+developing+pollen+cones+of+Norway+spruce+(Picea+abies)+are+homologous+to+the+B-class+floral+homeotic+genes+in+angiosperms.">MADS-box genes active in developing pollen cones of Norway spruce (Picea abies) are homologous to the B-class floral homeotic genes in angiosperms.</a> Dev Genet. 25 (3), 253-266 (1999).</li><li>Tandre, K., Svenson, M., Svensson, M. E., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+gene+structure+and+activity+in+the+regulation+of+reproductive+organ+development+of+conifers+and+angiosperms.">Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms.</a> Plant J. 15 (5), 615-623 (1998).</li><li>Winter, K. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+reveal+that+gnetophytes+are+more+closely+related+to+conifers+than+to+flowering+plants.">MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants.</a> Proc Natl Acad Sci U S A. 96 (13), 7342-7347 (1999).</li><li>Savard, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+and+nuclear+gene+sequences+indicate+late+Pennsylvanian+time+for+the+last+common+ancestor+of+extant+seed+plants.">Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.</a> Proc Natl Acad Sci U S A. 91 (11), 5163-5167 (1994).</li><li>Jack, T., Brockman, L. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeotic+gene+APETALA3+of+Arabidopsis+thaliana+encodes+a+MADS+box+and+is+expressed+in+petals+and+stamens.">The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens.</a> Cell. 68 (4), 683-697 (1992).</li><li>Bowman, J. L., Smyth, D. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+directing+flower+development+in+Arabidopsis.">Genes directing flower development in Arabidopsis.</a> Plant Cell. 1 (1), 37-52 (1989).</li><li>Kramer, E. M., Irish, V. 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The authors have nothing to disclose.

Two aspects of the P. abies protocol were optimized compared to the A. thaliana/B. napus protocol, RNase treatment and proteinase K concentration. The RNase removes background signals and thus increases the signal specificity 17. The proteinase K treatment is needed to permeabilize the tissues so the probe can enter and hybridize to the RNA pool of interest. The DAL13 antisense probe gave a higher signal without RNase-treatment (Fig. 3C, E, G) compared to sections treated with RNase for 30 min (Figure 2D, F and H). Since the RNase-treatment did not enhance the signal it was removed from the protocol. The proteinase K treatment were tested at three different concentrations; 1, 3 and 5 &micro;g/ml. In the case of P. abies a low or no signal was observed using 1 &micro;g/ml proteinase K (Fig. 3C-D). A strong signal was obtained with both 3 &micro;g/ml (Fig. 3E-F) and 5 &micro;g/ml (Fig. 3G-H) proteinase K. Since no apparent difference in signal strength was found when using 5 &micro;g/ml proteinase K compared to 3 &micro;g/ml, we chose to use 3 &micro;g/ml in our protocol. It is likely that the need of a higher proteinase K concentration in the P. abies male cones compared to the A. thaliana and B. napus floral buds is due to more compact tissue with higher levels of phenolics and polysaccharides.
During the fixation and embedding some differences between the A. thaliana/B. napus protocol and the P. abies protocol are evident. The tissue of interest is fixed to provide RNA retention and this is a critical step since poorly fixed materials might give a low or undetectable signal even though the mRNA is highly abundant. The tissue is dehydrated, cleared and embedded in wax in gradual changes to avoid tissue damage, and in some protocols 0.85% NaCl is added to the ethanol in the dehydration to further avoid shrinkage and swelling of the cells 3. The dehydration step during embedding can be performed on ice to keep RNase activity at minimum. In the P. abies protocol the 4% paraformaldehyde fix solution contains 0.25% glutaraldehyde, which is supposed to give a better RNA retention 17 even though that some argue that it probably make no difference 3. After sectioning the material is fixed onto slides and pretreated (i.e. dewaxed, rehydrated, permeabilized, treated to reduce non-specific binding of the probe and finally dehydrated) before the hybridization.
In the protocol presented here the whole detection procedure can be performed in ordinary laboratories, the signal develops usually within 1-3 days and the ratio between signal over background can continuously be monitored. The DIG-labeled probes usually give a more distinct signal as compared to radioactive labeled probes, are more stable and can be reused in consecutive experiments. On the other hand, the sensitivity is reduced compared to radioactive probes 17. In situ hybridization detects expression using a labeled probe specific for a certain mRNA. Radio-labeling is a robust and sensitive labeling method but it requires handling of radioactivity and it takes several weeks before the results can be detected. Antigen-labeled probes on the other hand, are more stable, less hazardous and require exposure to the detection medium for a few days only 17. Thus, antigen-labeling methods are currently dominating. The most critical step regardless of protocol is the probe design. Care should be taken to make sure the probe has a high specificity and is labeled with high quality UTPs. Sometimes the hybridization temperature and/or reaction length have to be adjusted for specific probes.
Our modified protocol based on DIG-labeled probes will facilitate localization of mRNA expression simultaneously in species ranging from A. thaliana to P. abies, and should with minor adjustments be usable in other plant species. The protocol has, beside male cones, also been tested on different stages of vegetative shoots and in both zygotic and somatic embryos from P. abies with good results (unpublished results; Karlgren et al.).

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<tr>
<td>Acetic anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6404-200ml</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Acrylamide, linear</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>9520</td>
<td>&nbsp;</td>
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<td>Anti-DIG-antibodies</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
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<td>&nbsp;</td>
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<td>Blocking reagent</td>
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<td>Roche Applied Science</td>
<td>11 175 041 910 or 11 096 176 001</td>
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<td>Bovine serum albumin</td>
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<td>Sigma-Aldrich</td>
<td>A7030-50g</td>
<td>minimum 98%</td>
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<td>Deionized formamide</td>
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<td>Sigma-Aldrich</td>
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<td>Roche Applied Science</td>
<td>11175025910</td>
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<td>Glutaraldehyde (25%)</td>
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<td>Histolab products AB</td>
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<td>Probe-on Plus slides</td>
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<td>Proteinase K</td>
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<td>P2308-5mg</td>
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<td>R5503-100mg</td>
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<td>Tissue-clear</td>
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<td>Sakura Finetek Europ&#233; BV</td>
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<td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td>
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<td>Triethanolamine</td>
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<td>Sigma-Aldrich</td>
<td>T1377-100ml</td>
<td>minimum 98%</td>
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<td>Triton X-100</td>
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<td>use your standard product in the lab</td>
<td>&nbsp;</td>
<td>Use one or both of Triton X-100 and Tween-20.</td>
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<td>tRNA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>83853-100mg</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>use your standard product in the lab</td>
<td>&nbsp;</td>
<td>Use one or both of Triton X-100 and Tween-20.</td>
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<td>Western Blue</td>
<td>&nbsp;</td>
<td>Promega Corp.</td>
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non-radioactive in situ hybridization protocol applicable for norway spruce and a range of plant species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Watch this Scientific Journal Video about Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species at JoVE.com

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

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20.9K Views.  Uppsala University. We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.