Name: Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques (Video) | JoVE Journal
Uploaded: 2018-04-12T13:00:00.000+00:00
Duration: 9 min 17 s
Description: Learn whole-mount clearing and staining techniques for dissecting Arabidopsis flower organs and siliques. This method enables rapid screening of developmental stages to study reproductive biology and plant development.

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5 minutes

This work was supported by the University of Zurich, an IEF Marie Curie Grant (grant no. TransEpigen-254797 to A.H.), an Advanced Grant of the European Research Council (grant no. MEDEA-250358 to U.G.), and a Research and Technology Development project (grant MecanX to U.G.) from SystemsX.ch, the Swiss Initiative in Systems Biology.

1. Flower and Silique Fixation
<ol>
	<li>Harvest flowers and siliques from plants synchronized at the opening of the first flower. 
	NOTE: Under the experimental conditions used here, plants start flowering approximately 21 days after transplantation from Murashige and Skoog (MS) plates to soil. Seeds are stratified for 3&#8211;4 days at 4 &#176;C and germinated/grown on MS plates at 22 &#176;C/16 h light and 18 &#176;C/8 h dark for 8 to 10 days, before transplanting the seedlings on nutrient-rich soil in pots kept under the same conditions (Figure 1). The number of replicates depends on the specific research objective, but a minimum of 5 inflorescences (from 5 individual plants) for each treatment is recommended. If flowers are the experimental unit, a minimum of 10 flowers per replicate is recommended.</li>
	<li>Cut inflorescences or flowers (Figure 1E-H) using small scissors (e.g., nail scissors) and place them immediately in a microcentrifuge tube (for single inflorescence/flower fixation) or a conical tube (for collective fixations) containing freshly made Carnoy&#39;s, methanol/acetic acid, or FPA50 fixatives (depending on the application, see below) on ice. 
	NOTE: Samples should be completely submerged in the fixative.</li>
	<li>Leave the tissue within the fixative for 4 h to overnight at 4 &#176;C. 
	NOTE: Vacuum infiltration can be used for speeding up the penetration of the fixative into the tissue, but this may be detrimental to preserving the structure of the tissue. The authors do not implement vacuum infiltration.</li>
	<li>Remove the fixative, add enough 70% ethanol to cover the samples, and return them to 4 &#176;C for at least 24 h; samples can stay indefinitely in this solution. After removing ethanol, proceed immediately to the next step; either dissection, or SDS clearing.</li>
</ol>
2. Dissection under the Stereomicroscope
<ol>
	<li>Put freshly made 70% ethanol in a watch maker&#39;s glass placed on a small Petri dish for support.</li>
	<li>Place the inflorescence/flower/silique on this freshly made fixative and dissect under the stereomicroscope using forceps and a syringe with a needle.
	<ol>
		<li>Use a watch maker&#39;s glass or similar device that allows the samples to be totally covered with ethanol (recommended) for the dissection of inflorescences, and for tearing apart floral organs of mature flowers (sepals, petals, and stamen can be dissected at this stage) to avoid the risk of samples drying out.</li>
	</ol>
	</li>
	<li>Dissect siliques and small flower buds on a slide as described below.
	<ol>
		<li>Put the watch maker&#39;s glass with pre-processed samples aside, and use a normal slide for the final dissection.</li>
		<li>Move the sample of interest to the slide and add 10 &#181;L of fresh 70% ethanol. Work swiftly on the final dissection and add 10 &#181;L of ethanol, if necessary, to keep the sample moist without excessive liquid.</li>
		<li>For releasing pollen grains from stamens, see step 5.1. For dissecting carpels and ovules, follow the procedure described in Figure 2. This procedure applies to all stages of development of the carpel, including the stage of development of green siliques. 
		NOTE: Do not add ethanol if there is sufficient ethanol carry-over from moving the samples; dissecting very small samples is much easier in a minimal amount of liquid. Always keep only the organs of interest (sepals, petals, stamens, carpels, ovules, seeds, or pollen grains) on the slide. This procedure will ensure a uniform thickness between the slide and coverslip, corresponding to the thickness of the organ under examination, resulting in a higher homogeneity, and thus efficiency for microscopic examinations (higher number of target specimens in the same focal plane).</li>
	</ol>
	</li>
	<li>Use a small piece of blotting paper to absorb and remove as much ethanol as possible. Quickly proceed to the next step (section 3, 4, or 6) before the sample dries out.</li>
</ol>
3. Chloral Hydrate-Based Clearing and Combined Clearing-staining
NOTE: Best results for chloral hydrate-based clearing are obtained with FPA50 fixed material.
<ol>
	<li>Place 20 &#181;L of clearing solution (chloral hydrate/glycerol, modified Hoyer&#39;s medium, Herr&#39;s 4&#189;, or Herr&#39;s IKI-4&#189; solutions) on the specimen-bearing slide. Using a pair of syringes with hypodermal needles, position the specimens as needed by reducing the space between them, and flipping those where the organ of interest faces downward. Remove any remaining air bubble using the needle.</li>
	<li>Lower a coverslip sideways and gently place it, pressing very gently, and wait until the clearing solution fills the space in between. Add minimal clearing solution, if needed, to fill the entire space under the coverslip.</li>
	<li>Place the slide on a slide holder and leave it under the fume hood. Proceed to microscopic observations after at least 4 h and during the following 4&#8211;5 days.</li>
</ol>
4. Combined Alexander Staining and Herr&#39;s 4&#189; Clearing of Anthers
NOTE: The original Alexander protocol is based on releasing pollen grains on the slide before staining. An efficient and more informative, modified Alexander staining and clearing procedure is the staining of mature pollen grains within mature but non-dehiscent anthers.
<ol>
	<li>Choose freshly harvested flower buds that are about to open with non-dehiscent anthers. 
	NOTE: Do not take younger flower buds because Alexander staining is intended for mature pollen grains and does not efficiently stain immature pollen grains. A minimum of 5 flowers per treatment harvested from different plants and inflorescences is recommended.</li>
	<li>Prepare a 96-well plate with enough Alexander&#39;s solution to submerge a flower bud. Expose the anthers by removing the sepals and petals and immerse them in Alexander&#39;s solution. Keep the samples under the fume hood for 1&#8211;3 h.</li>
	<li>Replace Alexander&#39;s solution with Herr&#39;s 4&#189; solution and place the multi-well plate back under the fume hood for an overnight incubation.</li>
	<li>Using forceps, gently move the cleared flower buds onto a new slide. Since some carry-over of Alexander&#39;s solution may occur, use a blotting paper to remove as much clearing solution as possible.</li>
	<li>Dissect the stamens and remove all other tissues. Follow the steps in section 3 using Herr&#39;s 4&#189; solution.</li>
</ol>
5. Removing the Exine from Pollen Grains
NOTE: We recommend using Carnoy&#39;s fixative for DAPI staining.
<ol>
	<li>Follow the fixation and dissection procedures described in sections 1 and 2. By now, one should have one to many stamens on the slide within a minimum amount of 70% ethanol.</li>
	<li>Use a blotting paper to remove excess ethanol, and add 5&#8211;10 &#181;L of DAPI solution. Using a couple of syringes with needles, release the pollen grains within that small amount of solution (e.g., use one syringe to immobilize the stamen and the other to release the pollen grains). Add another 5 &#181;L of DAPI if the solution dries out.</li>
	<li>Removing all debris of the stamen is a critical step, such that only pollen grains are left between the slide and the coverslip.</li>
	<li>Place a coverslip on the specimen-bearing slide by lowering it sideways and add the minimal amount of DAPI solution required to fill the space. Place an index finger on the coverslip, and without squashing too much make a quick, firm, and short sliding movement. 
	NOTE: To gauge the necessary force and amplitude of the sliding movement, this step should be practiced several times, checking the results each time under the microscope.</li>
	<li>Add DAPI solution if needed and place the slide in a humid box in the dark at 4 &#176;C for 15 min to 1 h. Proceed to observe samples under wide-field fluorescence or confocal microscopy within 24 h. Try to combine this procedure with SDS clearing to check whether further improvements can be obtained.</li>
</ol>
6. Sodium Dodecyl Sulfate (SDS) Clearing
NOTE: Depending on the floral organ to be analyzed, and on the researcher&#39;s skills to dissect very soft and small specimens, the SDS treatment can be carried out either before (for experienced researchers) or after the dissection step (for less experienced researchers) on methanol-acetic acid fixed material.
<ol>
	<li>Use a watch maker&#39;s glass, a concave slide, or a microcentrifuge tube to incubate dissected or non-dissected samples in 1% SDS and 0.2 N NaOH solution overnight at room temperature.</li>
	<li>Handle with care because the sample is very soft and has a transparent appearance. Rinse several times with water. 
	NOTE: Remnants of the SDS solution might lead to precipitation when adding the staining solution, e.g., aniline blue.</li>
	<li>For non-dissected samples, follow the dissection procedure described in section 2, using water instead of 70% ethanol. After dissecting the desired tissue, remove any remnants and debris, and any excess water with a blotting paper, then add 20&#8211;40 &#181;L of staining solution, and proceed with step 6.5.</li>
	<li>For dissected samples, carefully move the sample from the washing solution and place it onto a clean slide, and add 20&#8211;40 &#181;L of staining solution.</li>
	<li>Gently place a coverslip on the slide by lowering it sideways. Take care not to squash the preparation. If the tissue is too soft, place any thin separator in between the slide and the coverslip at its corners (e.g., tape or a broken coverslip), leaving a chamber-like space between slide and coverslip, such that the coverslip doesn&#39;t crush the specimen.</li>
	<li>Place all the slides within a humid box, cover it with aluminum foil, and place it at 4 &#176;C for at least 1 h. Proceed to observe the specimen using widefield fluorescence or confocal microscopy within 24 h.</li>
</ol>

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<li>Haseloff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Old+botanical+techniques+for+new+microscopes%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Old botanical techniques for new microscopes.</a> BioTechniques. 34 (6), 1174-1182 (2003).</li>
<li>Truernit, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+whole-mount+imaging+of+three-dimensional+tissue+organization+and+gene+expression+enables+the+study+of+phloem+development+and+structure+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis.</a> Plant Cell. 20 (6), 1494-1503 (2008).</li>
<li>Hedhly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Starch+turnover+and+metabolism+during+flower+and+early+embryo+development%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Starch turnover and metabolism during flower and early embryo development.</a> Plant Physiol. 172 (4), 2388-2402 (2016).</li>
<li>Crane, C. F., Carman, J. G. <a target="_blank" href="https://www.jstor.org/stable/2443827?seq=1#page_scan_tab_contents">Mechanisms of apomixis in Elymus rectisetus from eastern Australia and New Zealand.</a> Am J Bot. 74 (4), 477-496 (1987).</li>
<li>Young, B. A., Sherwood, R. T., Bashaw, E. C. <a target="_blank" href="http://www.nrcresearchpress.com/doi/abs/10.1139/b79-204">Cleared-pistil and thick-sectioning techniques for detecting aposporous apomixis in grasses.</a> Can J Bot. 57 (15), 1668-1672 (1979).</li>
<li>Kurihara, D., Mizuta, Y., Sato, Y., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ClearSee%3A+a+rapid+optical+clearing+reagent+for+whole-plant+fluorescence+imaging%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging.</a> Development. 142, 4168-4179 (2015).</li>
<li>Warner, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optical+clearing+technique+for+plant+tissues+allowing+deep+imaging+and+compatible+with+Fluorescence+Microscopy%5BTitle%5D)+AND+%22Plant+Phys%22%5BJournal%5D)">An optical clearing technique for plant tissues allowing deep imaging and compatible with Fluorescence Microscopy.</a> Plant Phys. 166, 1684-1687 (2014).</li>
<li>Hasegawa, J., Sakamoto, Y., Nakagami, S., Aida, M., Sawa, S., Matsunaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+imaging+of+plant+organs+using+a+simple+and+rapid+transparency+technique%5BTitle%5D)+AND+%22Plant+Cell+Physiol%22%5BJournal%5D)">Three-dimensional imaging of plant organs using a simple and rapid transparency technique.</a> Plant Cell Physiol. 57 (3), 462-472 (2016).</li>
<li>Musielak, T. J., Slane, D., Liebig, C., Bayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Versatile+Optical+Clearing+Protocol+for+Deep+Tissue+Imaging+of+Fluorescent+Proteins+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">A Versatile Optical Clearing Protocol for Deep Tissue Imaging of Fluorescent Proteins in Arabidopsis thaliana.</a> PLoS ONE. 11 (8), e0161107(2016).</li>
</ol>

The authors have nothing to disclose.

The existence of many flower buds within a single inflorescence of Arabidopsis, spanning all flower developmental stages, offers a unique opportunity for studies aimed at characterizing an effect of a treatment or a developmental feature simultaneously across the different stages of flower development. A good reference point between different individual plants is the opening of the first flower of the main inflorescence. Plants are treated in such a way that flowering is synchronized as much as possible (e.g...

Flowers are among the most important defining organs of angiosperms. Flowering plants appeared some 90&#8211;130 million years ago1, and diversified so fast that their rapid appearance was described as an &#34;abominable mystery&#34; by Charles Darwin2. The interests of plant researchers in flower development are diverse. Some research has focused on understanding the evolutionary origin of the flower as a whole, or the evolution of specific anatomical, structural, and functional properties of flowers3,4,5,6. The high variation in floral form and structure, as well as the modes of sexual and asexual reproduction relying on them, make the flower a highly complex structure. This has led to diverse efforts to characterize the anatomy and structural features of floral organs, using light and electron microscopical techniques that could be combined with genetic and molecular investigations7. Furthermore, as the source of fruits and seeds, flowers are of paramount importance for human and animal nutrition. Therefore, the characterization of flower and fruit development has many implications for applied research, including food security for an ever-increasing human population and ecological conservation strategies under a changing environment8,9,10.
Flower development in Arabidopsis starts with flower induction and the transformation of the vegetative meristem to an inflorescence (group of flowers) meristem. Flower primordia are initiated laterally on the flank of the inflorescence meristem11. The floral organ primordia form progressively in concentric whorls from the outside to the center of the flower, and eventually develop into sepals, petals, stamens, and carpels7. These floral organs fulfill distinct nutritive, protective, and functional (e.g., pollinator attraction) roles in different plant species, with the sexual organs sustaining the development of male and female gametophytes, respectively12,13. The gametophytes, in turn, each differentiate a pair of male (sperm) and female gametes (egg and central cell), which unite upon double fertilization to form the next generation, the zygote, and the primary endosperm, a terminal tissue supporting the development of the embryo14,15. Fruit and seed development support the growth, maturation, and preservation of the embryo and, eventually, its dispersal. Extensive research has been performed to characterize flower and embryo development in diverse plant species, especially in the model species Arabidopsis7,16,17.
Early microscopic analyses of flower development were based on time-consuming sample processing and observation techniques, such as paraffin or resin embedding and sectioning, combined with light or electron microscopy. These traditional microscopic techniques were often used in combination with molecular genetic investigations, such as microscopical analyses of mutants, the localization of RNA by in situ hybridization, or the immuno-detection of proteins. Recent progress in wide-field and confocal fluorescence microscopy, in state-of-the-art 3-D computer-aided image analyses, and the continuous refinement of molecular methods that can be used on minimally processed whole-mount specimens, has led to a need for efficient and minimal sample processing techniques that are preferentially amenable to quantitative analyses. In recent years, significant progress has been made in developing clearing techniques on whole-mount animal specimen. They render the sample transparent either by using aqueous urea- or sugar-based reagents (e.g., SCALE, SeeDB, CUBIC)18,19,20, or by selectively removing lipids (using the detergent SDS) after embedding samples in stable hydrogels; the removal of lipids can be achieved either by passive diffusion (e.g., modified CLARITY protocol21, PACT-PARS-RIMS22) or actively by electrophoresis (original CLARITY protocol23 and ACT-PRESTO24). Encouraged by this fast progress, some derived techniques are also emerging for use in plants.
In this methods paper focused on the model Arabidopsis, we describe the procedure for the proper dissection of flower buds, flowers, and young siliques, and the clearing of whole-mount samples for diverse staining and observation procedures using classical or a recent SDS-based clearing method. Examples for starch, callose, and chromatin staining are given. Although these procedures may need further improvements and adaptations when used with other species, we hope they will set the stage for further research on these simple but critical methods that are the starting point of many research projects.

<table><tbody><tr><td>&lt;strong&gt;Reagents and Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Scharlau</td><td>ET00102500</td><td></td></tr><tr><td>Acetic Acid</td><td>Applichem</td><td>A3686,2500</td><td>100% Molecular biology grade</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Molecular Biology Grade</td></tr><tr><td>Methanol</td><td>Scharlau</td><td>ME03062500</td><td></td></tr><tr><td>Formaldehyde Solution</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>Propionic acid</td><td>Sigma-Aldrich</td><td>81910-250 ml</td><td></td></tr><tr><td>Chloral hydrate</td><td>Sigma-Aldrich</td><td>15307</td><td></td></tr><tr><td>Glycerol</td><td>Roth</td><td>3783.1</td><td></td></tr><tr><td>Gum arabic</td><td>Fluka</td><td>51198</td><td></td></tr><tr><td>Lactic acid</td><td>Fluka</td><td>69773</td><td></td></tr><tr><td>Phenol</td><td>Sigma-Aldrich</td><td>77607-250ML</td><td>We used liquid phenol (use the density to find the required volume for your solution)</td></tr><tr><td>Clove oil</td><td>Sigma-Aldrich</td><td>C8392-100ML</td><td></td></tr><tr><td>Xylene</td><td>Roth</td><td>4436.1</td><td></td></tr><tr><td>Iodine</td><td>Fluka</td><td>57665</td><td></td></tr><tr><td>Potassium iodide</td><td>Merck</td><td>5043</td><td></td></tr><tr><td>Malachite Green</td><td>Fluka</td><td>63160</td><td></td></tr><tr><td>Fuchsin acid</td><td>Fluka</td><td>84600</td><td></td></tr><tr><td>Orange G</td><td>Sigma</td><td>7252</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>Molecular Biology Grade</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>71690</td><td></td></tr><tr><td>Sodium di-Hydrogen Phosphate</td><td>Applichem</td><td>A1047,1000</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S9763-1KG</td><td></td></tr><tr><td>Potassium phosphate</td><td>Sigma-Aldrich</td><td>04347</td><td></td></tr><tr><td>EDTA</td><td>Applichem</td><td>A2937,1000</td><td></td></tr><tr><td>Calcofluor</td><td>Sigma</td><td>F6259</td><td>Fluorescent brightener 28</td></tr><tr><td>Auramine</td><td>Chroma</td><td>10120</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>toxic</td></tr><tr><td>Triton-X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Aniline blue</td><td>Merck</td><td>1275</td><td></td></tr><tr><td>MS medium</td><td>Carolina</td><td>19-57030</td><td></td></tr><tr><td>Nutrient-rich substrate</td><td>Einheitserde</td><td>ED73</td><td></td></tr><tr><td>Watch maker&#039;s glass</td><td></td><td></td><td>No specific brand</td></tr><tr><td>15 ml falcon centrifuge tubes</td><td>VWR</td><td>62406-200</td><td></td></tr><tr><td>Dumond Forceps</td><td>Actimed</td><td>0208-5SPSF-PS</td><td></td></tr><tr><td>Forceps</td><td>DUMONT BIOLOGY</td><td>0108-5</td><td></td></tr><tr><td>Syringe</td><td>BD</td><td>BD Plastipak 300013</td><td>1 ml</td></tr><tr><td>Preparation needle</td><td>BD</td><td>BD Microlance 304000</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Scientific</td><td>10143562CE</td><td>cut edges</td></tr><tr><td>Coverslips</td><td>Thermo Scientific</td><td>DV40008</td><td></td></tr><tr><td>Humid box</td><td></td><td></td><td>A plastic box with damp paper towel and slide supports inside</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fixatives&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Carnoy&#039;s (Farmer&#039;s) fixative</td><td></td><td></td><td>Absolute ethanol : glacial acetic acid, 3:1 (ml:ml)</td></tr><tr><td>Methanol/acetic acid fixative</td><td></td><td></td><td>50 % (v/v) methanol, 10 % (v/v) glacial acetic acid in deionized water</td></tr><tr><td>FPA50 fixative</td><td></td><td></td><td>Formalin, propionic acid, 50% ethanol; 5:5:90 (ml:ml:ml)</td></tr><tr><td>&lt;strong&gt;Clearing solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chloral hydrate/glycerol</td><td></td><td></td><td>Chloral hydrate : glycerol : water, 8:1:2 (g:ml:ml). Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td></tr><tr><td>Modified Hoyer</td><td></td><td></td><td>Gum arabic 7.5 g, chloral hydrate 100 g, glycerol 5 ml , water 30 ml. Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td></tr><tr><td>Herr&#039;s 4&amp;frac12; clearing fluid</td><td></td><td></td><td>Lactic acid, chloral hydrate, phenol crystals, clove oil, xylene; 2:2:2:2:1, by weight. Can be used for all flower developmental stages (especially for stamen development) and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td></tr><tr><td>SDS/NaOH solution</td><td></td><td></td><td>Mix-dilute the the SDS and the NaOH stock solution to 1% SDS / 0.2 N NaOH (10x dilution). For all stages of flower and silique developmental stages. The best fixative is the methano/acetic acid fixative; the other two fixatives can also be used. Can be combined with calcofluor, auramine, DAPI, and aniline blue staining solution.</td></tr><tr><td>SDS stock solution</td><td></td><td></td><td>10 % (w/v) sodium dodecyl sulphate. Dissolve 10 g sodium dodecyl sulphate in 80 ml deionized water and make up to 100 ml with deionized water.</td></tr><tr><td>NaOH stock solution</td><td></td><td></td><td>2 N NaOH solution: dissolve 4 g of NaOH in 40 ml of deionized water and make up to 100 ml with deionized water</td></tr><tr><td>&lt;strong&gt;Combined clearing and staining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Herr&#039;s IKI-4&amp;frac12;</td><td></td><td></td><td>To a standard 4&amp;frac12; (9 g in total) add: 100 mg iodine, 500 mg potassium iodide. This clearing solution can be used for all flower developmental stages and for silique development, either for increasing contrast or for characterizing starch dynamics. Use FPA50 for structural analysis and Carnoy&#039;s fixative for quantitative starch analysis.</td></tr><tr><td>Alexander staining</td><td></td><td></td><td>Ethanol 95% 10ml, malachite green (1% in 95% EtOH) 1 ml, fuchsin acid (1% in ddH2O) 5ml, orange G (1% in ddH2O) 0.5ml, phenol 5g, chloral hydrate 5g, glacial acetic acid 2ml, glycerol 25ml . This clearing/staining alone or in combination with Herr&#039;s 4&amp;frac12; solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It&#039;s used on freshly harved non-fixed material.</td></tr><tr><td>&lt;strong&gt;Staining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcofluor solution</td><td></td><td></td><td>Calcofluor 0.007% in water (g:ml). Originally used as an optical brightner. Can be used for staining cellulose, carboxylated polysaccharides and callose in cell walls. Frequently used to stain the intine of the pollen grain. All three fixatives can be used with this solution.</td></tr><tr><td>Auramine solution</td><td></td><td></td><td>Auramine 0.01% in water (g:ml). This lipophilic fluorscent dye can be used for staining cuticles, cutin, and exine among others. All three fixatives can be used with this solution</td></tr><tr><td>Calcofluor-Auramine mixture</td><td></td><td></td><td>Auramine solution : Calcofluor solution, 3:1. Can be used for a combined staining by both solutions. Other proportions can be assayed maintaining a smaller proportion of calcofluor with respect to auramine.</td></tr><tr><td>DAPI solution</td><td></td><td></td><td>DAPI 0.4 ug/ml, 0.1 M sodium phosphate buffer (pH 7), 0.1% Triton-X-100, 1 mM EDTA. This solution can be used for staining chromosome spreads during male and female meiosis, and cell nuclei of any tissue. Frequently used for studying pollen grain development. Carnoy&#039;s and methanol/ acetic acid are the best fixatives for this solution. Formaldehyde-based fixatives such as FPA50 may interfere with the staining. Excitation in the UV and maximum emission around 461 nm.</td></tr><tr><td>Sodium phosphate buffer (0.1 M)</td><td></td><td></td><td>Proton receptor: 0.2 M Na2HPO4, proton donor: 0.2 M NaH2PO4, ratio proton donor / proton receptor: 1.364 ( for a pH 7)</td></tr><tr><td>Aniline blue solution</td><td></td><td></td><td>0.1% (w/v) aniline blue, 108 mM K3PO4 (pH 11), 2% glycerol. This solution can be used for staining callose and cellulose of many stages of development (e.g callose deposition in male and female terads, callose plugs in pollen tubes). Excitation in the UV and maximum emission around 455. It can also be excited at 514 nm with emission in the red for cell content staining.</td></tr></tbody></table>

whole-mount clearing and staining of arabidopsis flower organs and siliques

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.

Arabidopsis belongs to the Brassicacea family, bearing inflorescences with bisexual flowers arranged in a corymb (Figure 1). Each flower has four sepals, four petals, six stamens (four long and two short), and a syncarpous gynoecium consisting of two congenitally fused carpels (Figure 1F-H) arranged in four concentric whorls25,26. Arabidopsis...

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, ...

whole-mount-clearing-staining-arabidopsis-flower-organs

Research

JoVE Journal

Developmental Biology

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging.  Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

Live Confocal Imaging of Developing Arabidopsis Flowers

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

A Method for Characterizing Embryogenesis in Arabidopsis

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

Optical Clearing of Plant Tissues for Fluorescence Imaging

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana, which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Learn whole-mount clearing and staining techniques for dissecting Arabidopsis flower organs and siliques. This method enables rapid screening of developmental stages to study reproductive biology and plant development.

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

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