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>

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>Reagents and Materials</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&#39;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixatives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carnoy&#39;s (Farmer&#39;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>Clearing solutions</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&#39;s 4&#189; 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>Combined clearing and staining solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Herr&#39;s IKI-4&#189;</td>
			<td></td>
			<td></td>
			<td>To a standard 4&#189; (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&#39;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&#39;s 4&#189; solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It&#39;s used on freshly harved non-fixed material.</td>
		</tr>
		<tr>
			<td>Staining solutions</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&#39;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

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...

Watch this Scientific Journal Video about Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques at JoVE.com

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.

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, ...

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

Research

JoVE Journal

Developmental Biology

16.6K Views.  University of Zurich. 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.

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

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

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.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...