This work was supported by the USDA&#160;National Institute of Food and Agriculture, NRS Project No. FLA-TRC-006176 and the University of Florida Institute of Food and Agricultural Sciences.

1. Planting and pollination
NOTE: It is important to identify compatible genotypes whose hybridization would result in fruit set&#160;and the production of viable embryos.
<ol>
	<li>Planting conditions and maintenance
	<ol>
		<li>Obtain seeds of squash genotypes (cultivars/accessions) for hybridization (Table 1).</li>
		<li>Fill 50 cell starting flats (25 cm width x 50 cm length) with potting medium amended with complete NPK fertilizer containing 1.38 g/kg N, 1.38 g/kg P, and 1.38 g/kg K.</li>
		<li>Sow seeds to a depth equal to their length and cover with potting medium. Water the flats without creating standing water. Afterward, keep the media moist by watering it by hand once per day.</li>
		<li>At the second true-leaf stage, transplant the seedlings into 25 cm to 30 cm diameter pots amended with a complete NPK fertilizer at 3 tablespoons/pot. Fertilize the plants once a week with 500 mL/pot of liquid fertilizer containing NPK 20:20:20 added to a concentration of 1 g per gallon of water.</li>
		<li>Maintain plants in the greenhouse at temperatures between 22-28 &#176;C under natural light regime. For vining genotypes, provide a supporting trellis in the greenhouse (Figure 1).</li>
	</ol>
	</li>
	<li>Performing pollinations
	<ol>
		<li>Typically, flowering in squash starts at 6 to 8 weeks from seeding depending upon the cultivar (Figure 2A,B). Begin making controlled hybridization (crosses) as soon as the plants start flowering. Under greenhouse conditions, pollination can be done year-round.</li>
		<li>Identify male and female flowers of C. pepo and C. moschata cultivars that will be ready for pollination the following day. To identify such flowers, check for flowers whose petals have a yellow hue but are not open. Gently tape the flowers closed at the top using masking tape to prevent accidental insect pollination (Figure 2C,D).</li>
		<li>The morning of the following day, the flowers are ready to pollinate. Carry out pollination before 10:00 a.m. to improve the rate of hybridization success27.</li>
		<li>Open the female and male flowers by gently removing the taped top portion of the petals. Remove the petals from the male flower and transfer the pollen by gently rubbing the anther on the stigma of the female flower (Figure 3A).</li>
		<li>After pollination, immediately close the pollinated female flower with masking tape. Use a tag to record the date of pollination and to indicate the paternal and maternal parents used in the cross (Figure 3B).</li>
		<li>A successful cross is indicated by an expanded ovary which quickly forms a small fruit within 1 week (Figure 4A). The plant is then ready for harvest 45-55 days after pollination28 (Figure 4B).</li>
	</ol>
	</li>
</ol>
2. Embryo rescue technique
<ol>
	<li>Media preparation
	<ol>
		<li>Prepare antibiotic stocks: For cefotaxime (sodium salt), dissolve the antibiotic in 4 mL of deionized or distilled water, filter through a sterile 0.22 &#181;m syringe filter, and make 0.5 mL aliquots. Store&#160;at -20&#160;&#176;C. The resulting stock solution will have a concentration of 250 mg/mL.</li>
		<li>For ampicillin (sodium salt), dissolve the powder in 10 mL of water, filter through a sterile 0.22 &#181;m syringe filter, and aliquot in 1 mL stock with a final concentration of 100 mg/mL. Store&#160;at -20&#160;&#176;C.</li>
		<li>Make Murashige and Skoog (MS) medium by dissolving 2.45 g of medium (4.91&#160;g/L concentration) in 500 mL of distilled water in a 1 L bottle. Add 1.5 g of gellan gum&#160;and autoclave at 121 &#176;C for 20 min. The gellan gum completely dissolves during autoclaving.</li>
		<li>After autoclaving, cool the medium by placing the bottle in a water bath at 50 &#176;C. Remove the antibiotic stocks from the freezer and thaw them in the laminar flow cabinet.</li>
		<li>Transfer the medium bottle to the laminar flow hood. Add 0.6 mL of cefotaxime stock solution (250 mg/mL)&#160;and 0.25 mL of ampicillin stock (100 mg/mL) to the medium bottle and mix well. Pour about 7 mL of the medium into a sterile Petri dish (60 mm x 15 mm).</li>
		<li>Allow the medium to solidify in the Petri dishes for approximately 15-20 min. Close the Petri dishes with sealing wrap and place them into a storage box. Store at room temperature.</li>
	</ol>
	</li>
	<li>Embryo rescue
	<ol>
		<li>Before starting, clean and sterilize the laminar-air-flow cabinet with 70% ethyl alcohol.</li>
		<li>Harvest the squash fruit by breaking/cutting it off the main vine and disinfect the fruit surface by washing with liquid detergent (e.g., 0.3% chloroxylenol) in the lab sink until all the loose dirt is removed (Figure 5A).</li>
		<li>Rinse with ample tap water. Dry the fruit with clean paper towels. Move the fruit to the laminar-air-flow cabinet (Figure 5B).</li>
		<li>Surface sterilize the fruit by spraying 70% ethanol on the fruit in the sterile laminar-air-flow cabinet. Bisect the fruit with a sterile knife (Figure 5C) and extract the seeds.</li>
		<li>Use sterile forceps to aseptically open the seed coat and expose the immature embryos (Figure 6). Carefully place the immature embryos in a Petri dish containing MS medium supplemented with cefotaxime and ampicillin (Figure 7A). Close the Petri dish and seal with wrapping film. 
		NOTE: Depending on the size of the embryo, it is possible to fit five or six embryos in a Petri dish.</li>
		<li>Place the sealed Petri dishes with embryos in a growth chamber under 16 h photoperiod at 25 &#176;C and 70% relative humidity. If contamination occurs, promptly subculture the uncontaminated embryos into a new plate with the same medium.</li>
		<li>The cotyledons will start expanding after 4 days and will turn green in 10 days (Figure 7B). At this point, if needed, perform subculturing into new plates containing the same medium to allow tissue expansion. The roots will start to appear at 14 days (Figure 7C), and at 21 days the plantlets will have extended roots and cotyledons (Figure 7D). 
		NOTE: The culture medium used in the protocol is appropriate for differentiation into shoots and roots without supplementary growth regulators.</li>
		<li>At this stage, remove the plantlets from the&#160;Petri dishes and gently wash off the media from the roots with tap water (Figure 8). Place the plantlets in a plastic container (14 cm x 9 cm x 4 cm) and cover the roots with wet paper towels (Figure 9A). Cover the container and remoisten the paper towels as necessary.</li>
		<li>Keep the containers at room temperature (25-28 &#176;C) and with a 16 h photoperiod. This step will acclimatize the plantlets. After acclimatization for 7-10 days in the container, the seedlings will be approximately 3-4 in long. During this period, remoisten the paper towels as needed.</li>
		<li>Transfer the seedlings into 50 cell starting flats (25 cm width x 50 cm length) amended with fertilizer as previously described and move them to the greenhouse (Figure 9B). Do not over-water seedlings to avoid rotting; add approximately 10-20 mL per cell as needed.</li>
		<li>At the second to third true-leaf stage, transplant the seedlings into 30 cm diameter pots filled with potting medium amended with fertilizer as previously described (Figure 10A). Provide trellis support for vining plants and perform controlled hybridization when the plants start flowering, as previously described (Figure 10B).</li>
		<li>Maintain the plants in the greenhouse at temperatures between 22-28 &#176;C under natural light regime. Evaluate the plants for fruit and seed characteristics.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

There are two main bottlenecks for successful interspecific hybridization between C. moschata and C. pepo: cross-compatibility barrier, which is determined by genotype responsiveness to produce hybrid embryos, and post-fertilization barriers, which hinder the development of hybrid embryos to normal seeds. As previously reported for squash, the cross-compatibility test in the current study revealed that most of the fruit developed parthenocarpically, with most of the seeds unviable16</s...

Cucurbita (2n = 40) is a highly diverse genus in the Cucurbitaceae family that contains 27 different species, of which five are domesticated1. Among these, Cucurbita moschata, C. pepo, and C. maxima are the most economically important worldwide. In the U.S., C. moschata and C. pepo are the two most important species in agricultural production. C. pepo consists of four subspecies (ovifera, pepo, fraternal, and gumala) that contain both summer and winter squash cultivar groups of crookneck, straightneck, acorn, scallop, cocozelle, vegetable marrow, zucchini, and pumpkin2,3,4,5. C. moschata primarily consists of winter squash market types including butternut, Dickinson, and cheese group1. The two species are morphologically and phenotypically diverse, with C. pepo regarded for its yield, earliness, bush growth habit, and diverse fruit traits including fruit shape, fruit size, flesh color, and rind pattern. On the other hand, C. moschata is prized for its adaptation to heat and humidity, as well as disease and pest resistance6,7. Interspecific hybridization between C. moschata and C. pepo is not only an important strategy for introgression of desirable characteristics between the two species, but also allows for the broadening of the genetic base in breeding programs7,8.
Early crosses between C. moschata and C. pepo were made to determine their compatibility and/or taxonomic barriers9,10,11, whereas later studies mostly focused on transferring desirable traits12,13,14. Interspecific hybridization between the two species has targeted the transfer of novel traits such as a bush or semi-bush growth habit and improved yield from C. pepo along with disease resistance, adaptability to abiotic stress, and increased vigor from C. moschata14,15,16. For example, specific crosses between C. pepo (P5) and C. moschata (MO3) have resulted in higher fruit yield13, while C. moschata accessions (Nigerian Local and Menina) have been widely used as the primary source of resistance to potyviruses in cultivated C. pepo cultivars17,18.
Previous studies showed that hybridization between C. moschata and C. pepo is possible but difficult8,15. The interspecific crosses could result in no fruit set (abortion), parthenocarpic fruits devoid of viable seeds (empty seeds), seedless fruits where the immature embryos fail to develop (stenospermocarpy), or fruits with few immature embryos that can be rescued into mature plants through embryo rescue15,16. For instance, no viable seeds were obtained by crossing C. pepo (table queen, maternal) with C. moschata (large cheese, paternal), however, the reciprocal cross yielded 57 viable seeds from 134 pollinations9. Hayase obtained viable seeds from C. moschata and C. pepo crosses only when crosses were made at 04:00 a.m. using pollen stored at 10 &#176;C overnight19. Baggett crossed eight different C. moschata varieties with C. pepo (delicata) and reported that out of 103 total pollinations, 83 fruits were obtained that appeared normal, but none of them contained viable seeds8. In a cross between C. pepo (S179) and C. moschata (NK), Zhang et al. obtained 15 fruits with 2,994 seeds, but only 12 of those seeds were viable while the remaining displayed only rudimentary development. These studies suggest that even though interspecific crossing between C. moschata and C. pepo is highly beneficial, obtaining fruits with viable seeds from the crosses is demanding16.
Embryo rescue has been suggested as an appropriate method to overcome problems arising from early aborting or poorly developed embryos and is one of the earliest and most successful in vitro culture techniques for regeneration of immature embryos16,20. Embryo rescue involves the in vitro culture of under-developed/immature embryos followed by transfer to a sterile nutrient medium to facilitate recovery of seedlings and ultimately mature plants21. Although embryo rescue is commonly used in squash breeding, a detailed description of the appropriate methodology that would allow its routine application is lacking. Using embryo rescue technique to overcome interspecific hybridization barriers in Cucurbita species was reported as early as 195422. However, the success of embryo rescue in the early studies was either unreported or very low. Metwally et al. reported a 10% success rate (regeneration into mature plants) among 100 interspecific hybrid embryos rescued from a cross between C. pepo and C. martinezii23. Sisko et al. reported a variable success rate of embryo regeneration among embryos obtained from different cross combinations: the regeneration rate of hybrids obtained by crossing C. maxima (Bos. Max)and C. pepo (Gold Rush) was 15.5%, for C. pepo (Zucchini) and C. moschata (Hokaido) was 20%, while for C. pepo (Gold Rush) and C. moschata (Dolga) it was 37.5%24. In addition to genotype, media and in vitro culture conditions are important factors for the success of the technique25,26. In the current study, various cross combinations between C. moschata and C. pepo were&#160;tested, and a simple methodology for utilizing the embryo rescue technique in squash was developed. The development of a simple and easily reproducible embryo rescue technique will facilitate interspecific hybridization and germplasm enhancement in squash breeding programs.

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			<td>NPK fertilizer (20-20-20)</td>
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			<td>BWI Companie,s Inc</td>
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			<td>USA Scientific, Inc</td>
			<td>8609-0160</td>
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			<td>BWI Companies, Inc</td>
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			<td>plastic food containers, reused</td>
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			<td>BWI Companies Inc</td>
			<td>GPPF128S4</td>
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			<td>syringe filter (0.22 um )</td>
			<td>ExtraGene</td>
			<td>B25CA022-S</td>
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			<td>trellis support</td>
			<td>The Home Depot</td>
			<td>&#160;2A060006</td>
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			<td>water bath</td>
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embryo rescue protocol for interspecific hybridization in squash

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature embryos generated from these wide crosses must be regenerated using appropriate embryo rescue techniques. Although this technique is well established for many crops, a detailed description of the appropriate methodology for squash that would allow its routine application is lacking. Here, we describe an embryo rescue protocol useful for interspecific hybridization of C. pepo and C. moschata. To identify viable combinations for embryo rescue, 24 interspecific crosses were performed. Fruit set was obtained from twenty-two crosses, indicating a 92% success rate. However, most of the fruits obtained were parthenocarpic, with seeds devoid of embryos (empty seeds). Only one cross combination contained immature embryos that could be regenerated using basal plant growth media. A total of 10 embryos were rescued from the interspecific F1 fruit, and the success rate of embryo rescue was 80%. The embryo rescue protocol developed here will be useful for interspecific hybridization in squash breeding programs.

Fruit set and seed viability 
An initial test was conducted to determine fruit set and seed viability in a variety of cross-combinations. A total of 15 squash genotypes, four C. pepo and 11 C. moschata, were chosen (Table 1). Out of the 24 interspecific cross combinations attempted, a fruit set was obtained for 22 (Table 2), representing an overall &#62;92% success in the fruit set. No mature fruits were obtained by crossing O and M and E and J, wh...

Watch this Scientific Journal Video about Embryo Rescue Protocol for Interspecific Hybridization in Squash at JoVE.com

Embryo Rescue Protocol for Interspecific Hybridization in Squash

The&#160;article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature ...

embryo-rescue-protocol-for-interspecific-hybridization-in-squash

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2.5K Views.  UF/IFAS Tropical Research and Education Center. The article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

Video: Embryo Rescue Protocol for Interspecific Hybridization in Squash

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

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

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

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

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

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...