This research was funded by Ministerio de Ciencia, Innovaci&#243;n y Universidades-European Regional Development Fund, European Union (AGL2016-77267-R, and AGL2015-74071-JIN); Instituto Nacional de Investigaci&#243;n y Tecnolog&#237;a Agraria y Alimentaria (RFP2015-00015-00, RTA2017-00003-00); Gobierno de Arag&#243;n-European Social Fund, European Union (Grupo Consolidado A12_17R), Fundaci&#243;n Biodiversidad, and Agroseguro S.A.

1. Self-(in)compatibility determination
<ol>
	<li>Sample the flowers in the field. It is necessary to collect the flowers at balloon stage (Figure 1A), corresponding to stage 58 on the BBCH scale for apricot28, to avoid unwanted previous pollination.</li>
	<li>Self- and cross-pollinations in the laboratory
	<ol>
		<li>Remove the anthers of the flowers at balloon stage and place them on a piece of paper to dry at laboratory temperature.</li>
		<li>After 24 h, sieve the pollen grains by using a fine mesh (0.26 mm) (Figure 1B).</li>
		<li>Emasculate a group of 30 flowers at the same balloon stage for each self-pollination and cross-pollination and place the pistils on florist foam in water at laboratory temperature (Figure 1C).</li>
		<li>Hand pollinate the pistils with the help of a paintbrush with pollen from flowers of the same cultivar 24 h after emasculation. In addition, pollinate another set of pistils of each cultivar with pollen from flowers of a compatible pollinizer as control (Figure 1D).</li>
		<li>After 72 h, fix the pistils in a fixative solution of ethanol/acetic acid (3:1) for at least 24 h at 4 &#176;C29. Then discard the fixative and add 75% ethanol ensuring that the samples are completely submerged in the solution. Samples can be conserved in this solution at 4 &#176;C until use8,17,30,31,32.</li>
	</ol>
	</li>
	<li>Evaluating pollen viability through in vitro pollen germination
	<ol>
		<li>To prepare the germination medium, weight 25 g of sucrose, 0.075 g of boric acid (H3BO3) and 0.075 g of calcium nitrate (Ca(NO3)2)33.</li>
		<li>Add the components of the medium in 250 mL of distilled water and dissolve completely.</li>
		<li>Solidify the medium adding 2 g of agarose and mix by swirling.</li>
		<li>Check the pH of the medium using a pH meter and adjust the value to 7.0 with NaOH or HCl solution.</li>
		<li>Autoclave the mixture to sterilize the medium.</li>
		<li>After autoclaving, cool down the medium and distribute it into Petri dishes in a sterile laminar flow hood.</li>
		<li>Scatter the pollen grains of the same cultivars used for the controlled pollinations in the solidified pollen germination medium and observe them under the microscope after 24 h6. 
		NOTE: To sterilize the laminar flow hood, clean the surface with 70% ethanol and switch on the UV lamp during 10 min.</li>
		<li>Store the Petri dishes in a refrigerator at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Microscopy observations
	<ol>
		<li>Wash the pistils three times for 1 h with distilled water and leave them in 5% sodium sulphite at 4 &#176;C. After 24 h, autoclave them at 1 kg/cm2 during 10 min in sodium sulphite to soften the tissues34.</li>
		<li>Place the autoclaved pistils over a glass slide and, with the help of a scalpel, remove the trichomes around the ovary to get a better visualization of the pollen tubes. Then, squash the pistils with a cover glass.</li>
		<li>Prepare 0.1% (v/v) aniline blue stain: mix 0.1 mL of aniline blue in 100 mL of 0.1 N potassium phosphate tribasic (K3PO4). Apply a drop of aniline blue over the preparations to stain callose depositions during pollen tube growth.</li>
		<li>Observe the pollen tubes along the style by a microscope with UV epifluorescence using 340-380 bandpass and 425 longpass filters.</li>
	</ol>
	</li>
</ol>
2. DNA extraction
<ol>
	<li>Sample 2-3 leaves in the field in spring. It is recommended to sample the leaves at young stages since DNA obtained is of higher quality and lower levels of phenolic compounds compared to old leaves.</li>
	<li>Extract Genomic DNA following the steps described in a commercially available kit (see Table of Materials).</li>
	<li>Analyze the quantity and quality of DNA concentrations using UV-vis spectrophotometer (260 nm).</li>
</ol>
3. S-allele identification
<ol>
	<li>Setting up of the PCR Reactions
	<ol>
		<li>Prepare a 50 ng/&#956;L dilution in distilled water of each DNA extraction sample.</li>
		<li>Thaw out the PCR reagents slowly and keep them on ice. Leave the DNA polymerase in the freezer until needed.</li>
		<li>Prepare the amplification reactions using the different combinations of primers. Create the PCR reaction mix by combining the components in Table 1. Vortex the PCR reaction mix well and distribute the volume indicated for the different combinations of primers to each well of the PCR plate. Then, add 1 &#956;L of the DNA dilution in each well.</li>
		<li>Place the PCR plate in the thermocycler and run the corresponding PCR program shown in Table 1.</li>
	</ol>
	</li>
	<li>Analyze the amplified fragments. There are mainly two different ways to analyze the PCR amplified fragments: capillary electrophoresis (CE) with fluorescent-labelled primers or as visualize amplicons of agarose gel electrophoresis with not-labelled primers.
	<ol>
		<li>Capillary Electrophoresis
		<ol>
			<li>To prepare the loading buffer, mix 35 &#956;L of deionized formamide with 0.45 &#956;L of labeled sizing standard. Vortex the reagent to mix well, and then dispense 35.5 &#956;L into the well of the reader plate.</li>
			<li>Add 1 &#956;L of the PCR product into the well. In addition, add a drop of mineral oil to prevent water evaporation.</li>
			<li>Prepare the separation plate adding separation buffer.</li>
			<li>Use the commercial software included with the gene analyzer (see Table of Materials). Create a new sample plate and save the sample names for all wells on the plate.</li>
			<li>Select the method of analysis. In this case, denature the samples at 90 &#176;C for 120 s, inject at 2.0 kV for 30 s, and separate at 6.0 kV for 35 min.</li>
			<li>Insert the two plates into the gene analyzer. Fill the capillary array with distilled water.</li>
			<li>Load the patented linear polyacrylamide (LPA) gel. Finally, click Run.</li>
		</ol>
		</li>
		<li>Gel Electrophoresis
		<ol>
			<li>Prepare a 1% agarose gel adding 1.5 g of molecular biology grade agarose in 150 mL of 1x TAE (Tris-acetate-EDTA) electrophoresis running buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA at pH 8.0). Dissolve the agarose by microwave heating for 2-3 min.</li>
			<li>To visualize the DNA, add 4 &#956;L of a nucleic acid stain (see Table of Materials) and mix gently.</li>
			<li>Add a gel comb, with sufficient wells for ladders, controls and samples, into a gel tray. Then, pour slowly the mix into the middle of the gel tray and avoid bubbles.</li>
			<li>Let the gel cool down for 30-45 min at room temperature until the gel has completely solidified. Introduce the gel in the electrophoresis chamber, remove the gel comb and fill the chamber with enough 1x TAE buffer to cover the gel. 
			NOTE: Check the placement of the gel. The wells should be placed close to the negative pole since negatively charged DNA migrates towards the cathode.</li>
			<li>Add 5 &#956;L of loading buffer (0.1% (v/v) bromophenol blue) to the PCR products and mix well.</li>
			<li>To estimate the size of the bands, load 5 &#956;L of DNA molecular weight ladder (see Table of Materials).</li>
			<li>Load the samples into the additional wells of the gel.</li>
			<li>Once all the samples and the DNA molecular weight ladder are loaded, run the gel at 90 V for 1-1.5 h, until the blue dye line is approximately at 75% the length of the gel.</li>
			<li>Visualize the bands in a transilluminator for nucleic acids.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Traditionally, most commercial apricot European cultivars were self-compatible36. Nevertheless, the use of North American self-incompatible cultivars as parents in breeding programs in the last decades has resulted in the release of an increasing number of new self-incompatible cultivars with unknown pollination requirements7,8,37. Thus, the determination of self- and inter-(in)compatibility relationships...

Self-incompatibility is a strategy of flowering plants to prevent self-pollination and promote outcrossing1. In Rosaceae, this mechanism is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S2. In the style, the RNase gene encodes the S-stylar determinant, a RNase3, while a F-box protein, which determines the S-pollen determinant, is codified by the SFB gene4. The self-incompatibility interaction takes place through the inhibition of pollen tube growth along the style preventing the fertilization of the ovule5,6.
In apricot, a varietal renewal has taken place worldwide in the last two decades7,8. This introduction of an important number of new cultivars, from different public and private breeding programs, has resulted in the increase of apricot cultivars with unknown pollination requirements8.
Different methodologies have been used to determine pollination requirements in apricot. In the field, self-(in)compatibility may be established by controlled pollinations in caged trees or in emasculated flowers and subsequently recording the percentage of fruit set9,10,11,12. In addition, controlled pollinations have been carried out in the laboratory by semi-in vivo culture of flowers and analysis of the pollen tube behavior under fluorescence microscopy8,13,14,15,16,17. Recently, molecular techniques, such as PCR analysis and sequencing, have allowed the characterization of incompatibility relationships based on the study of the RNase and SFB genes18,19. In apricot, thirty-three S-alleles have been reported (S1 to S20, S22 to S30, S52, S53, Sv, Sx), including one allele related with self-compatibility (Sc)12,18,20,21,22,23,24. Up to now, 26 incompatibility groups have been stablished in this species according to the S-genotype8,9,17,25,26,27. Cultivars with the same S-alleles are inter-incompatible, whereas cultivars with at least one different S-allele and, consequently, allocated in different incompatible groups, are inter-compatible.
To define the pollination requirements of apricot cultivars, we describe a methodology that combines the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis in apricot cultivars. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars.

<table>
	<tbody>
		<tr>
			<td>Agarose D1 Low EEO</td>
			<td>Conda</td>
			<td>8010.22</td>
			<td></td>
		</tr>
		<tr>
			<td>BIOTAQ DNA Polymerase kit</td>
			<td>Bioline</td>
			<td>BIO-21060</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field microscope</td>
			<td>Leica Microsystems</td>
			<td>DM2500</td>
			<td></td>
		</tr>
		<tr>
			<td>CEQ System Software</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>QIAGEN</td>
			<td>69106</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set, 4 x 25 &#181;mol</td>
			<td>Bioline</td>
			<td>BIO-39025</td>
			<td></td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit - 400</td>
			<td>Beckman Coulter</td>
			<td>608098</td>
			<td></td>
		</tr>
		<tr>
			<td>GenomeLab GeXP Genetic Analysis System</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GenomeLab Separation Buffer</td>
			<td>Beckman Coulter</td>
			<td>608012</td>
			<td></td>
		</tr>
		<tr>
			<td>GenomeLab Separation Gel LPA-1</td>
			<td>Beckman Coulter</td>
			<td>391438</td>
			<td></td>
		</tr>
		<tr>
			<td>HyperLadder 100bp</td>
			<td>Bioline</td>
			<td>BIO-33029</td>
			<td></td>
		</tr>
		<tr>
			<td>HyperLadder 1kb</td>
			<td>Bioline</td>
			<td>BIO-33025</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis System</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Imager VersaDoc MP 4000 system&#160;</td>
			<td>Bio-Rad</td>
			<td>170-8640</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-400-518</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-Meter BASIC 20</td>
			<td>Crison</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion&#160;High-Fidelity PCR Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>F553S</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Pack P 25 T</td>
			<td>Biometra</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer Forward</td>
			<td>Isogen Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer Reverse</td>
			<td>Isogen Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quantity One Software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope</td>
			<td>Leica Microsystems</td>
			<td>MZ-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell GT</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>QIAGEN</td>
			<td>201203</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Stand Autoclave</td>
			<td>JP Selecta</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of self- and inter-(in)compatibility relationships in apricot combining hand-pollination, microscopy and genetic analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Pollination studies in apricot require the use of flowers at the late balloon stage one day before anthesis (Figure 1A). This stage is considered the most favorable for both pollen and pistil collection, since floral structures are nearly mature, but anther dehiscence has not yet occurred. This prevents the interference of undesired pollen, not only of pollen from the same flower but also from other flowers, since the closed petals impede the arrival of insects carrying exte...

Watch this Scientific Journal Video about Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses at JoVE.com

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...

determination-self-inter-compatibility-relationships-apricot

Research

JoVE Journal

Developmental Biology

7.2K Views.  Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA). We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Video: Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

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 ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...