Name: fluorescent immunolocalization of arabinogalactan proteins and pectins in the cell wall of plant tissues
Uploaded: 2021-02-27T13:00:00
Description: Watch this Scientific Journal Video about Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues at JoVE.com

The authors received support from the EU project 690946 &#8216;SexSeed&#8217; (Sexual Plant Reproduction &#8211; Seed Formation) funded by H2020-MSCA-RISE-2015 and SeedWheels FCT PTDC/BIA-FBT/27839/2017. AMP received a grant from the European Union&#8217;s MSCA-IF-2016 project (no. 753328). MC received a grant from FCT PhD grant SFRH/BD/111781/2015.

1. Sample Preparation: fixation, dehydration, and LR-White embedding
<ol>
	<li>Fixation and dehydration 
	NOTE: The fixation process is critical to preserve the sample; by crosslinking molecules the cellular metabolism is stopped, ensuring cellular integrity and preventing molecular diffusion. Fixative agents and the concentration used must be adjusted to that purpose, leaving the antigens sufficiently exposed to interact with the antibodies. The following protocol combines the mild fixative capab...

<ol>
	<li>Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+Cell+Walls.">Plant Cell Walls.</a> Plant Physiology. 154 (2), 483-486 (2010).</li><li>Pereira, A. M., Lopes, A. L., Coimbra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan+Proteins+as+Interactors+along+the+Crosstalk+between+the+Pollen+Tube+and+the+Female+Tissues.">Arabinogalactan Proteins as Interactors along the Crosstalk between the Pollen Tube and the Female Tissues.</a> Frontiers in Plant Science. 7 (7), (2016).</li><li>Showalter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan-proteins:+structure,+expression+and+function.">Arabinogalactan-proteins: structure, expression and function.</a> Cellular and Molecular Life Sciences. 58 (10), 1399-1417 (2001).</li><li>Majewska-Sawka, A., Nothnagel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+roles+of+arabinogalactan+proteins+in+plant+development.">The multiple roles of arabinogalactan proteins in plant development.</a> Plant Physiology. 122, 3-9 (2000).</li><li>Seifert, G., Roberts, 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+biology+of+arabinogalactan+proteins.">The biology of arabinogalactan proteins.</a> Annual Review of Plant Biology. 58, 137-161 (2007).</li><li>Ruprecht, C., 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=A+Synthetic+Glycan+Microarray+Enables+Epitope+Mapping+of+Plant+Cell+Wall+Glycan-Directed+Antibodies.">A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.</a> Plant Physiology. 175 (3), 1094-1104 (2017).</li><li>Verhertbruggen, Y., Walker, J. L., Guillon, F., Scheller, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparative+Study+of+Sample+Preparation+for+Staining+and+Immunodetection+of+Plant+Cell+Walls+by+Light+Microscopy.">A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy.</a> Frontiers in Plant Science. 29 (8), 1505 (2017).</li><li>Osborn, M., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+and+immunocytochemical+procedures+with+affinity+purified+antibodies:+tubulin+containing+structures.">Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin containing structures.</a> Methods in Cell Biology. 24, 97-132 (1982).</li><li>Coimbra, S., Almeida, J., Junqueira, V., Costa, M., Pereira, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan+proteins+as+molecular+markers+in+Arabidopsis+thaliana+sexual+reproduction.">Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction.</a> Journal of Experimental Botany. 58 (15), 4027-4035 (2007).</li><li>Wilson, S. M., Bacic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+plant+cells+for+transmission+electron+microscopy+to+optimize+immunogold+labeling+of+carbohydrate+and+protein+epitopes.">Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes.</a> Nature Protocols. 7 (9), 1716-1727 (2012).</li><li>Gane, A., Clarke, A., Bacic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+expression+of+arabinogalactan-proteins+in+the+ovaries+of+Nicotiana+alata+Link+and+Otto.">Localization and expression of arabinogalactan-proteins in the ovaries of Nicotiana alata Link and Otto.</a> Sex Plant Reproduction. 8, 278-282 (1995).</li><li>Coimbra, S., Salema, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+arabinogalactan+proteins+in+Amaranthus+hypocondriacus+L.+ovules.">Immunolocalization of arabinogalactan proteins in Amaranthus hypocondriacus L. ovules.</a> Protoplasma. 199 (1-2), 75-82 (1997).</li><li>Coimbra, S., Duarte, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan+proteins+may+facilitate+the+movement+of+pollen+tubes+from+the+stigma+to+the+ovules+in+Actinidia+deliciosa+and+Amaranthus+hypocondriacus.">Arabinogalactan proteins may facilitate the movement of pollen tubes from the stigma to the ovules in Actinidia deliciosa and Amaranthus hypocondriacus.</a> Euphytica. 133 (2), 171-178 (2003).</li><li>El-Tantawy, A., 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=Arabinogalactan+protein+profiles+and+distribution+patterns+during+microspore+embryogenesis+and+pollen+development+in+Brassica+napus.">Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus.</a> Plant Reproduction. 26 (3), 231-243 (2013).</li><li>Costa, M., Pereira, A. M., Rudall, P. J., Coimbra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+arabinogalactan+proteins+(AGPs)+in+reproductive+structures+of+an+early-divergent+angiosperm,+Trithuria+submersa+(Hydatellaceae).">Immunolocalization of arabinogalactan proteins (AGPs) in reproductive structures of an early-divergent angiosperm, Trithuria submersa (Hydatellaceae).</a> Annals of Botany. 111 (2), 183-190 (2013).</li><li>Costa, M., Sobral, R., Ribeiro Costa, M. M., Amorim, M. I., Coimbra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+presence+of+arabinogalactan+proteins+and+pectins+during+Quercus+suber+male+gametogenesis.">Evaluation of the presence of arabinogalactan proteins and pectins during Quercus suber male gametogenesis.</a> Annals of Botany. 115 (1), 81-92 (2015).</li><li>Hibbs, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Immunolabelling.">Fluorescence Immunolabelling.</a> Confocal Microscopy for Biologists. , (2004).</li><li>Johnson, G. D., 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=Fading+of+Immunofluorescence+during+microscopy:+A+Study+of+the+Phenomenon+and+its+Remedy.">Fading of Immunofluorescence during microscopy: A Study of the Phenomenon and its Remedy.</a> Journal of Immunological Methods. 55 (2), 231-242 (1982).</li><li>Baird, T. R., Kaufman, D., Brown, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+Free+Microscopy:+An+Opportunity+for+Core+Facility+Directors.">Mercury Free Microscopy: An Opportunity for Core Facility Directors.</a> Journal of Biomolecular Techniques. 25 (2), 48-53 (2014).</li><li>Weber, G. F., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+image+acquisition+using+a+monochrome+camera+and+standard+fluorescence+filter+cubes.">Color image acquisition using a monochrome camera and standard fluorescence filter cubes.</a> BioTechniques. 38 (1), 52-56 (2005).</li></ol>

The fluorescent immunolocalization method in plants here described, while seamlessly straightforward, relies on the success of several small steps. The first of which is sample preparation and fixation. During this first step, a mixture of formaldehyde and glutaraldehyde is used to crosslink the majority of the cell components. The formaldehyde in the solution provides a mild and reversible fixation while the glutaraldehyde provides a strong more permanent linkage; the balance between the two fixatives provides the appro...

Plant cell walls are complex structures composed of polysaccharides and glycoproteins. Cell walls are extremely dynamic structures whose architecture, organization and composition vary according to cell type, localization, developmental stage, external and internal stimuli1. Arabinogalactan proteins (AGPs) and pectins are important components of the plant cell wall. AGPs are highly glycosylated proteins and pectins are homogalacturonan polysaccharides whose composition, amount and structure vary greatly during different plant developmental stages2,3,4. AGPs and pectin studies have revealed their involvement in several plant processes such as programmed cell death, response to abiotic stresses, sexual plant reproduction, among many others5. Most of these studies started with information obtained from immunolocalization studies.
Given its complexity, the study of cell walls requires many different tools. Detection of glycan epitopes using monoclonal antibodies (mAbs) is a valuable approach to resolve polysaccharide and glycoprotein distribution along this structure. There is a large collection of mAbs available to detect glycan epitopes and the specificity of each mAb is continuously being improved as well6. The technique here described is applicable to all plant species, and is a perfect tool to guide future research directions that might involve more expensive and complex techniques.
In this technique, specific antibodies are chemically conjugated to fluorescent dyes such as FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine-5-(and 6)-isothiocyanate) or several Alexa Fluor dyes. Immunofluorescence offers several advantages, allowing a clear and quick subcellular localization of glycans that can be directly observed under a fluorescence microscope. It is highly specific and sensitive, since the preparation of the sample can effectively protect the natural structure of the antigen, even if present in lower amounts. It allows the detection of multiple antigens in the same sample and most important, offers high quality and visually beautiful results. Despite the great power offered by fluorescence immunolocalization studies, they are often regarded as difficult to perform and implement most probably due to the lack of detailed protocols allowing the visualization of the different steps of the procedure. Here, we provide some simple guidelines on how to perform this technique and how to obtain high quality images.
For the protocol presented here, samples must first be fixed and embedded using the most appropriate fixative. Although considered as a time consuming and relatively tedious technique, proper fixation and embedding of the plant sample is the key to ensure a successful immunolocalization assay. For this purpose, the most usual is chemical fixation using crosslinking fixatives, like aldehydes. Cross-linking fixatives establish chemical bonds between molecules of the tissue, stabilizing and hardening the sample. Formaldehyde and glutaraldehyde are cross-linking fixatives, and sometimes a mix of both fixatives is used7. Formaldehyde offers great structural preservation of tissues and for extended periods of time, producing small tissue retractions and being compatible with immunostaining. Glutaraldehyde is a stronger and stable fixative usually used in combination with formaldehyde. The use of glutaraldehyde has some disadvantages that must be taken into account as it introduces some free aldehyde groups into the fixed tissue, which may generate some unspecific labeling. Also the crosslinking between proteins and other molecules occasionally may render some target epitopes inaccessible for the antibodies. To avoid this, the quantity and duration of the fixation must be carefully defined.
After fixation, samples are embedded in the proper resin to harden before obtaining the sections. London Resin (LR-White) acrylic resin is the resin of choice for immunolocalization studies. Unlike other resins, LR-White is hydrophilic, allowing the antibodies to reach their antigens, with no need of any treatment to facilitate it. LR-White has also the advantage of offering low auto-fluorescence, allowing a reduction in background noise during immunofluorescence imaging.
There are many staining techniques available to detect different components of the cell wall, such as Alcian blue staining, toluidine blue staining or Periodic acid&#8211;Schiff (PAS) staining. None of these offers the power of immunolocalization analyses8. This approach gives greater specificity in the detection of glycans, offering vaster information regarding cell wall composition and structure.

<table border="0" cellpadding="0" cellspacing="0" style="width:728px;" width="728">
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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">25% (w/v) Gluteraldehyde</td>
			<td style="width:99px;">Agar Scientific</td>
			<td style="width:196px;">AGR1010</td>
			<td style="width:187px;">aq. Solution, methanol free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">8 wells Glass reaction slides</td>
			<td style="width:99px;">Marinfeld</td>
			<td style="width:196px;">MARI1216750</td>
			<td style="width:187px;">other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acetic acid</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">A6283</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anti-Rat IgG (wole molecule)-FITC antibody produce in GOAT</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">F6258</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">cover slips, 24 mm x 50 mm</td>
			<td style="width:99px;">Marinfeld</td>
			<td>MARI0100222</td>
			<td style="width:187px;">The cover slip should cover all the wells. Other brands may be used&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:247px;">ddH2O</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol absolute</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fluorescent brightner 28</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">F-6259</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Gelatin capsules</td>
			<td style="width:99px;">Agar scientific</td>
			<td>AGG29211</td>
			<td style="width:187px;">The capsule size sould feat the size of the sample.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:247px;">Glass vials</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">Any simple unexpensive glass vials that can be sealed, may be used. The vials may be clean with 96% etanol after use to remove LR-White residue and reused.&#160;</td>
		</tr>
		<tr height="294">
			<td height="294" style="height:294px;width:247px;">LR-white medium grade, embdeding resin</td>
			<td style="width:99px;">Agar Scientific</td>
			<td>AGR1281</td>
			<td style="width:187px;">LR-White comes in several forms the medium grade provides na adequate cutting suport for most tissues for harder tissues a harder grade of LR-white may be recomendable. If possible use a resin already mixed with the polimeration activator (benzoyl peroxide), if not please folow the instructions of the suplier to prepare the resin.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Non fat dry milk</td>
			<td style="width:99px;">Nestl&#233;</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">any non fat dry milk is adequate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Oven</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">generic laboratory oven</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Petri dish, 10 cm x 10 cm square</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PIPES</td>
			<td style="width:99px;">Sigma Aldrich</td>
			<td style="width:196px;">P1851</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="357">
			<td height="357" style="height:357px;width:247px;">Rat generated Monoclonal Anti-Body</td>
			<td style="width:99px;">Plant probes</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">Several antibodies that recognize cell wall components are 
			available at both the Complex Carbohydrate research center 
			(CCRC, Georgia University USA) and Plant Probes (Paul 
			Knox Cell Wall Lab, at Leeds University UK). A short list of 
			some commonly used MABS and where they can be purchased 
			is presented in Supplemental Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Razor blades</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">regular razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">SDS</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">L6026</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Toluidine Blue-O</td>
			<td style="width:99px;">Agar Scientific</td>
			<td>AGR1727</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tween 20</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">P9416</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Ultramicrotome</td>
			<td style="width:99px;">Leica Microsystems</td>
			<td style="width:196px;">UC7</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">upright epifluorescence microscope with UV and FITC fluorescence filters</td>
			<td style="width:99px;">Leica Mycrosistems</td>
			<td style="width:196px;">DMLb</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">vaccum chamber</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">vaccum pump</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="297">
			<td height="297" style="height:297px;width:247px;">Vectashield</td>
			<td style="width:99px;">vecta Labs</td>
			<td style="width:196px;">T-1000</td>
			<td style="width:187px;">Other anti-fade may be used. Please do check for compatibility with FITC and the Fluorescente brightner 28. (Note: for a non-commercial alternative, (see Jonhson et al 198218) An antifade medium can be made by mixing 25 mg/mL of 1,4-Diazobicyclo-(2,2,2)octane (DABCO) in 9:1 (v/v) glycerol to 1xPBS. Adjust pH to 8.6 with diluted HCl.)</td>
		</tr>
	</tbody>
</table>

fluorescent immunolocalization of arabinogalactan proteins and pectins in the cell wall of plant tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

In a successful experiment, the secondary antibody will specifically pinpoint the location of the specific epitope in bright green, in a consistent manner, allowing for the characterization of the cell wall composition at a certain development stage of the cell, tissue or organ. For example the LM6 antibody has an high affinity for 1,5-arabinan, a compound with type-I rhamnogalacturonan that can be found abundantly labelling the cell wall of the developing Quercus suber anther (Figure 2A</st...

Watch this Scientific Journal Video about Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues at JoVE.com

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

The amount of information from only one experiment is enormous. We can detect the presence of these cell wall polymers and know exactly how they are distributed in cells and tissues, and even determine their abundance. This method is designed in such a way that it can be applied to almost any kind of plant tissue from any species one may want to analyze.Immunolocalization in plant tissues involves a lot of meticulous work, difficult to explain without actually seeing what happens. Before collecting the plant tissue sample, fill a glass vial with enough cold fixative solution to completely submerge all the samples. Then select the plant tissues to be analyzed and trim the samples to a size of no more than 16 square millimeters.Immediately immerse the samples in the fixative solution. Glutaraldehyde and formaldehyde are both excellent fixatives, but both are hazardous and must be handled in the flow hood. Transfer the vial to a vacuum chamber and slowly apply vacuum.When the pressure reaches negative 60 kilo pascals, the floating material in the vial will start to sink to the bottom. Maintain the pressure in the chamber at no more than negative 80 kilo pascals. The sinking process may take up to two hours.After the samples have been in the vacuum chamber for two hours, slowly release the vacuum. Seal the glass vial and refrigerate it overnight at four degrees Celsius. After the overnight fixation, discard any samples that didn't sink to the bottom of the vial.Wash the remaining samples with 25 millimolar PBS for 10 minutes and then wash them in 25 millimolar PIPES buffer for 20 minutes. It is extremely important to remove all floating samples because it's highly probable that the fixative has not penetrated the sample. Dehydrate the samples in an ethanol series, immersing the samples for 20 minutes at each ethanol concentration.To perfuse the samples, add increasing concentrations of LR-white resin in ethanol, beginning with one part resin to three parts ethanol. At each concentration, incubate for 24 hours at four degrees Celsius. Replace the LR-white resin with fresh resin and incubate the samples for an additional 12 hours at four degrees Celsius.Prepare the embedding gelatin capsules, selecting capsules that are slightly larger than the samples so that the samples can be completely enclosed in the resin. Label paper tags with pencil because ink will contaminate the resin. Apply one drop of fresh LR-white resin to the bottom of each capsule.Place a sample in each capsule, then fill the capsules to maximum capacity with fresh resin. Put the cap on the capsule and press gently to seal it. To polymerize the resin, cure the capsules at 58 degrees Celsius until fully hardened around 24 to 48 hours.After peeling the gelatin capsule from the LR-white resin, place the resin block under the stereo microscope. Using a sharp razor blade, trim the LR-white block at a 45 degree angle to the sample. Rotate the sample and make a second cut at a 90 degree angle to the first.Continue cutting in the same pattern to form a pyramid with the apex of the pyramid directly above the sample's area of interest, then begin removing fine slices perpendicular to the major axis until the cutting surface reaches the sample. The target sample should ideally be enclosed in a trapezoid shape. Place the trimmed resin block in the ultra microtome.Adjust the angle between the knife edge and the resin block. Cut sections from the block with a thickness between 200 and 700 nanometers. Use a wire loop to carefully lift some sampled sections from the microtome.Place them in a drop of distilled deionized water on a glass slide. Evaporate the water by placing the slide on a hot plate at 50 degrees Celsius, then add a drop of stain to the sections. After 30 seconds, rinse off the stain and observe the slide under the microscope to verify the general state of the section and that the desired plant structure is visible.Prepare a clean reaction slide by placing a drop of distilled deionized water in each well of the slide. Transfer one or two sample sections to each drop of water. Place the slide in a 10 centimeter square Petri dish, cover it and let it dry at 50 degrees Celsius.To prepare an incubation chamber for the reaction slides, place some dampened paper towels at the bottom of a pipette tips box and wrap the box with aluminum foil. Transfer the slides from the box to the incubation chamber. Pipette 50 microliters of blocking solution into each well.After incubating the slide for 10 minutes, remove the blocking solution, then wash all the wells twice with PBS for 10 minutes each time. After preparing the primary antibody solutions as described in the manuscript, perform a final wash of the wells with distill deionized water for five minutes. Pipette the primary antibody solution into the reaction wells, then pipette the blocking solution into the control wells.Close the incubation chamber. Let it stand for two hours at room temperature and then refrigerate it overnight at four degrees Celsius. Prepare a 1%solution of the secondary antibody in blocking solution.Approximately 40 microliters per well will be needed. Cover the solution with aluminum foil. Wash the wells of the reaction slide twice with PBS and once with distilled deionized water for 10 minutes each time.Ensure that no blocking solution or deposits remain in the wells. Pipette the secondary antibody solution into all the wells. Incubate the slides in the dark at room temperature for three to four hours.Again, wash the wells of the reaction slide twice with PBS and once with distilled deionized water, then add a drop of Calcofluor White to each well. Without washing, add a drop of mounting medium to each well and cover each well with a coverslip. Observe the sample sections in the reaction slide using a fluorescence microscope.For each observation, use a UV filter to detect cell walls stained by the Calcofluor and a FITC filter to detect immunolocalization. For a better visualization of the results, use ImageJ or a similar image analysis program to merge each UV filter image with the corresponding FITC filter image. By pinpointing the localization of specific epitopes, the protocol enables characterization of the cell wall composition.For example, 1, 5-arabinan is abundant in the cell wall of the developing Quercus suber anther. Scarcely esterified homogalacturonans are typically found at the root tip of Quercus suber embryo specifying mechanical properties of the organ. Xylogalacturonans are found in degenerating cells, such as the endosperm cell during the final stages of the Quercus suber acorn maturation.AGP's epitopes recognized by JIM13 or JIM8 are found on structures related to reproduction, such as cell lines related to microgametogenesis in Arabidopsis thaliana and the stigmatic papillae and microbial of the basal angiosperm Trithuria submersa. Common mistakes in implementing this protocol are usually easy to detect and identify. When the washes are skipped or the reaction wells are allowed to dry, the secondary antibody will usually appear as a smear.Aggregates of green fluorochrome will form if the unbound primary antibody is not properly washed away. Folding or detachment of the sections is usually related to poor adhesion due to the use of unclean slides or aggressive washing. Sample preparation is also critical.The resin blocks should be free of cracks with a clearly visible pale yellow to light brown sample. Inefficiently embedded samples will show powdery white spots or areas. Keeping the sample size under eight millimeters is essential for penetration of the fixative solution.Poorly fixed samples will appear dark brown or almost black. Excessive temperature can cause the resin to crack making sectioning of the sample impossible. The use of immunolocalization techniques open several research directions.One can follow on with more specific techniques such as biochemical analysis of the cell wall or even proceed to a molecular approach. The results provided by this technique can help to understand the composition of the cell wall, an extremely complex structure very difficult to analyze by simple chemical analysis.

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Research

JoVE Journal

Developmental Biology

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

This protocol describes applications of sample immobilization using Fibrin clots, limiting drifting, and allowing the addition and washout of reagents during live imaging. Samples are transferred to a drop of Fibrinogen containing culture medium on the surface of a coverslip, after which polymerization is induced by adding thrombin.

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...