This work was supported by National Key Research and Development Program (Grant number: 2017YFD0200600), the earmarked fund for China Agriculture Research System (grant number: CARS-23-D07) and the Bill &#38; Melinda Gates Foundation (Investment ID OPP1149777). We thank Prof. Jian-Xiang Wu for providing TYLCV CP antibodies.

1. Whitefly, Virus, Plants, and Acquisition of the Virus
<ol>
	<li>Rear whiteflies (MEAM1) on cotton (Gossypium hirsutum cv. Zhemian 1793) in insect-proof cages in a greenhouse at 26 &#177; 1 &#176;C with a 14:10 light:dark cycle and 60 &#177; 10% relative humidity.</li>
	<li>Perform conventional PCR based on whitefly mitochondrial cytochrome oxidase I gene to determine the purity of the whitefly population.
	<ol>
		<li>Collect 20 adult whiteflies and transfer them individually to a PCR tube containing 30 &#956;L of lysis buffer (10 mM Tris, pH = 8.4, 50 mM KCl, 0.45% [wt/vol] Tween-20, 0.2% [wt/vol] gelatin, 0.45% [vol/vol] Nonidet P 40, 60 g/mL Proteinase K).</li>
		<li>Add 5&#8211;7 ceramic beads (2 mm in diameter) into each PCR tube and grind the samples to perform the tissue lysis.</li>
		<li>Incubate all samples at 65 &#176;C for 1 h followed by 10 min at 100 &#176;C, and then centrifuge briefly. Use this supernatant as the template for PCR amplification. 
		NOTE: The incubation time at 65 &#176;C can be increased if necessary.</li>
		<li>Prepare the PCR reaction as follows: 1 U of Taq DNA polymerase, 2 &#181;L of 10x buffer (with Mg2+), 1.6 &#181;L of dNTP mixture (2.5 mM), 0.5 &#181;L of each primer (10 &#956;M each), 2 &#181;L of whitefly tissue lysate supernatant, and double distilled water to a total volume of 20 &#181;L per reaction. The forward primer sequence is 5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3&#39; and the reverse primer sequence is 5&#39;-TAATATGGCAGATTAGTGCATTGGA-3&#39;. 
		NOTE: A negative control where the DNA is substituted with nuclease-free water and a positive control with previously verified MEAM1 whitefly DNA should be included.</li>
		<li>Perform PCR using the following cycling parameters: initial denaturation at 95 &#176;C for 3 min, followed by 35 cycles of denaturation at 95 &#176;C for 30 s, annealing at 50&#8211;55 &#176;C for 30 s, and extension at 72 &#176;C for 1 min, followed by 72 &#176;C for 10 min.</li>
		<li>Digest the amplified product with restriction enzyme Taq I. To do so, prepare the digestion mixture with 0.1 &#181;L (1 U) of enzyme,1 &#956;L of 10x Taq I Buffer, 1 &#956;L of BSA, 5 &#956;L of amplified product, and double distilled water to a total volume of 10 &#956;L. Incubate at 65 &#176;C for 1 h in a thermocycler.</li>
		<li>Perform agarose gel electrophoresis of the samples by loading 5&#8211;7 &#181;L of each digested product and a DNA ladder (100-2,000 bp) into the wells of a 1% agarose gel. Then, observe the DNA band sizes by gel-red staining in a gel documentation machine using UV light. Compare the band sizes of the digested products to the positive control to determine the purity of the whitefly population.</li>
	</ol>
	</li>
	<li>Agro-inoculate the infectious clone of TYLCV (GenBank accession number AM282874.1) (pBINPLUS-SH2-1.4A) into 3&#8211;4 true leaf stage tomato plants (Solanum lycopersicum L. cv. Hezuo 903).
	<ol>
		<li>Inoculate 10 mL of Luria&#8211;Bertani (LB) medium containing kanamycin (50 &#956;g/mL) and rifampicin (50 &#956;g/mL) with a single colony. Incubate at 28 &#176;C with shaking at 200 rpm until the OD600 reaches ~1.5.</li>
		<li>Harvest agrobacteria by centrifugation at 4,000 x g for 10 min. Discard the supernatant.</li>
		<li>Resuspend pellets in 10 mL of infiltration buffer, consisting of 200 &#956;M acetosyringone, 10 mM 2-(N-morpholino) ethane sulfonic acid (MES), and 10 mM MgCl2. Incubate at room temperature (RT) for 1&#8211;2 h.</li>
		<li>Infiltrate 2&#8211;3 plant leaves using the mixture from step 1.3.3 with a 1 mL needleless syringe. Maintain plants in a greenhouse at 26 &#177; 1 &#176;C.</li>
	</ol>
	</li>
	<li>About 1 month later, perform a visual inspection and choose plants showing yellow curl leaf and stunt symptoms.</li>
	<li>Transfer non-viruliferous whitefly adults onto TYLCV-infected tomato plants for 48 h for virus acquisition.</li>
</ol>
2. Dissection and Collection of Midguts, Primary Salivary Glands (PSG), Ovaries, and Hemolymph of Female Whiteflies
<ol>
	<li>Collect whiteflies using aspiration, and then place them on ice to put them in a coma. Add 1x PBS (phosphate-buffered saline) buffer onto a microscope slide, and then place whiteflies in the 1x PBS buffer for dissection under a stereo microscope.
	<ol>
		<li>For the midgut dissection, break the abdomen of the whitefly and pull out the midgut using a fine acupuncture needle (0.25 mm in diameter). Transfer the midgut to clean, 1x PBS.</li>
		<li>For PSG dissection, break the body of the whitefly in the thorax near the head from the dorsal side. Next, insert a needle into the thorax to fix the whitefly, and use another needle to shake the whitefly until the two salivary glands are separated from the body. Then, transfer the PSGs to clean, 1x PBS.</li>
		<li>For the ovary dissection, completely break the abdomen of the whitefly, and use a needle to separate the ovaries from other tissues. Then, remove excess tissues by pipetting.</li>
		<li>For the hemolymph collection, add 10 &#956;L of 1x PBS buffer onto a microscope slide, and then place a whitefly in the buffer. To obtain the hemolymph, gently tear a hole in the abdomen using a fine acupuncture needle, and slightly press the abdomen to release the hemolymph out into the buffer. Collect 8 &#956;L of the liquid as the hemolymph sample.</li>
	</ol>
	</li>
</ol>
3. Localization of TYLCV in Whitefly Midgut, Primary Salivary Glands, and Ovaries by Immunofluorescence
NOTE: The procedure for the localization of TYLCV in the midguts is presented as an example. The method can be used for PSGs and ovaries.
<ol>
	<li>Dissect the midguts in TBS buffer (10 mM Tris-HCl, 150 mM sodium chloride, pH = 7.5) as described in step 2.2.1. Collect 15&#8211;20 whitefly midguts, transfer them into a glass Petri dish (3.5 cm in diameter), and then remove excess TBS buffer.</li>
	<li>Add 1 mL of 4% paraformaldehyde to fix midguts for 2 h at room temperature, and then remove the fixative solution. Pipette slowly to wash the midguts 3x in TBS for 2 min each.</li>
	<li>Incubate the midguts in 1 mL of 0.5% Triton X-100 for 30 min to permeabilize. Then remove the permeabilization solution and wash the midguts 3x with TBST (1x TBS with 0.05% Tween 20) as described in step 3.2.</li>
	<li>Add 1 mL of 1% BSA (bovine serum albumin prepared in TBST) to block the midguts. Perform the blocking step at RT for 2 h, and then remove the blocking solution and wash the midguts 3x in TBST.</li>
	<li>Dilute the primary antibodies (MAb 1C4 against TYLCV coat protein) at 1:400 with TBST24 and add the solution into the Petri dish to immerse the midguts. Incubate at 4 &#176;C overnight in the dark. Discard the primary antibody solution and wash 3x in TBST.</li>
	<li>Dilute the secondary antibodies (anti-mouse) at 1:400 with TBST and add the solution into the Petri dish to immerse the midguts. Incubate the dish for 2 h at RT in the dark. Discard the secondary antibody solution and wash the midguts 3x in TBST.</li>
	<li>Transfer the midguts to a clear microscope slide. Aspirate as much TBST off the slide as possible. Add 1 drop of DAPI (4&#39;, 6-diamidino-2-phenylindole, dihydrochloride) to stain the nucleus, and then place the coverslip and seal with nail polish. Examine under a confocal microscope.</li>
</ol>
4. Quantification of TYLCV in Whitefly Midgut, Primary Salivary Glands, Hemolymph, and Ovaries with qPCR
<ol>
	<li>Dissect the whitefly midguts, PSGs, hemolymph, and ovaries in 1x PBS as described above.</li>
	<li>Extract the DNA of the midguts, PSGs, hemolymph, and ovaries.
	<ol>
		<li>After dissection, wash the midguts, PSGs, and ovaries in clean, 1x PBS buffer 2&#8211;3x.</li>
		<li>Collect 20 midguts or 20 PSGs in 5 &#956;L of 1x PBS and transfer them into 25 &#956;L lysis solution. Transfer each ovary in 5 &#956;L of 1x PBS and then transfer into 25 &#956;L lysis solution individually.</li>
		<li>Grind midguts, PSGs, and ovaries samples as described in step 1.2.2.</li>
		<li>Collect 8 &#956;L of hemolymph with 1x PBS into a PCR tube, and then add 10 &#956;L of lysis solution. Mix thoroughly.</li>
	</ol>
	</li>
	<li>Extract DNA from all samples as described in 1.2.3.</li>
	<li>Prepare two separate qPCR master mixes (18 &#956;L each) for TYLCV and the internal control (&#946;-actin gene) as follows: 10 &#956;L of SYBR Green mix, 0.8 &#956;L of each primer (10 &#956;M each), and 6.4 &#956;L of double distilled water per reaction. For TYLCV, the forward primer sequence is 5&#8242;-GAAGCGACCAGGCGATATAA-3&#8242; and the reverse primer sequence is 5&#8242;-GGAACATCAGGGCTTCGATA-3&#8242;. For &#946;-actin, the forward primer sequence is 5&#8242;-TCTTCCAGCCATCCTTCTTG-3&#8242; and the reverse primer sequence is 5&#8242;-CGGTGATTTCCTTCTGCATT-3&#8242;.</li>
	<li>Dispense 18 &#181;L of the master mix into vials of qPCR strip tubes or a 96 well plate. Then, add 2 &#181;L of the DNA samples into each vial. Perform all reactions with at least three technical replicates and three biological replicates. 
	NOTE: Step 4.5 should be performed on ice.</li>
	<li>Close the qPCR tubes or seal the 96 well plates with adhesive plate seal.</li>
	<li>Centrifuge to collect the liquid at the bottom of the qPCR tubes or 96 well plate.</li>
	<li>Place the tubes or plate into the real-time thermal cycler and perform qPCR.
	<ol>
		<li>Perform the qPCR using the following cycling parameters: 95 &#176;C for 30 s, followed by 40 cycles of 95 &#176;C for 5 s and 60 &#176;C for 34 s for primer annealing and elongation, 1 cycle at 95 &#176;C for 15 s, ending with a melting curve step of 60 &#176;C to 95 &#176;C with an increment of 0.5 &#176;C per 15 s.</li>
		<li>Choose SYBR as the fluorophore dye and unknown as sample type.</li>
	</ol>
	</li>
	<li>Export the quantitative data to a spreadsheet format and analyze the relative quantity of viral DNA by the 2&#8722;&#916;&#916;CT method28.</li>
</ol>

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The authors have nothing to disclose.

Here we describe a protocol for the localization and quantification of a begomovirus in the tissues of its whitefly vector by immunofluorescence and qPCR. Dissection represents the first step to localize and quantify the virus in whitefly tissues. The whitefly body is about 1 mm in length, which means that the tissues are extremely small, and it is difficult to dissect them. Besides, there are strong connections between the tissues. For example, the ovaries are tightly connected with the bacteriocytes, making them diffic...

In the last decades, begomoviruses (genus Begomovirus, family Geminiviridae) have caused serious damage to the production of many vegetable, fiber, and ornamental crops worldwide1. Begomoviruses are transmitted in a persistent manner by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), which is a complex species containing over 35 cryptic species2,3. Begomoviruses may directly or indirectly affect whitefly physiology and behavior, such as fecundity4, longevity4, and host preference5,6. Furthermore, the transmission efficiency of a given begomovirus species/strain varies for different whitefly cryptic species even under the same experimental conditions7,8,9,10, indicating that there is a complex interaction between begomoviruses and whiteflies. To better understand the mechanisms underlying whitefly-begomovirus interactions, localization and quantification of the virus in whitefly tissues are essential.
Tomato yellow leaf curl virus (TYLCV) is a begomovirus that was first reported in Israel but nowadays causes serious damage to tomato production worldwide11,12. Due to its economic importance, it is one of the best-studied begomoviruses13. Like other monopartite begomoviruses, TYLCV is a single-strand circular DNA virus with a genome size of about 2,800 nucleotides14. While still under debate, several lines of evidence support the replication of TYLCV in whiteflies15,16,17. Moreover, the interaction of TYLCV particles and whitefly proteins has been reported6,18,19,20. For virus transmission, whiteflies acquire TYLCV by feeding on virus-infected plants, virions pass along the food canal to reach the esophagus, penetrate the midgut wall to reach the hemolymph, and then translocate into the primary salivary glands (PSGs). Finally, virions are egested with saliva along the salivary duct into plant phloem21. Furthermore, several studies show that TYLCV is able to be transovarially transmitted from female whiteflies to their offspring22,23. In other words, to achieve productive transmission, the virus has to overcome cellular barriers within the whitefly to translocate from one tissue to another. During the crossing of these barriers, interactions between whitefly and virus proteins are likely to occur, probably determining the efficiency with which the viruses are transmitted.
Immunofluorescence is a commonly used technique for protein distribution analysis. The specificity of antibodies binding to their antigen form the basis of immunofluorescence. Due to the economic significance of TYLCV, monoclonal antibodies against the TYLCV coat protein have been developed, offering a highly sensitive way to localize the virus24. Quantitative PCR (qPCR) allows sensitive and specific quantification of nucleic acids. This technique is most frequently based on the use of hydrolysis probe (e.g., TaqMan) or fluorescent dye (e.g., SYBR Green) detection. For hydrolysis probe-based qPCR, specific probes are needed, which consequently increase the cost. Fluorescent dye-based qPCR is simpler and more cost-effective, because labeled amplicon-specific hybridization probes are not required25. So far, several studies have used immunofluorescence and qPCR along with other methods to investigate the complex begomovirus-whitefly interactions. For example, Pan et al. performed qPCR and immunofluorescence analysis of the virus in whitefly tissues and found that the difference in ability to transmit tobacco curly shoot virus (TbCSV) between whitefly species AsiaII 1 and Middle East Asia Minor 1 (MEAM1) was due to the virus being able to efficiently cross the midgut wall of AsiaII1 but not MEAM18. Similarly, while Mediterranean (MED) whiteflies can readily transmit TYLCV, they fail to transmit tomato yellow leaf curl China virus (TYLCCNV). Selective transmission was investigated using immunofluorescence detection of virus in the PSGs, which showed that TYLCCNV do not easily cross the PSGs of MED whiteflies26. Immunofluorescence colocalization of TYLCV CP and the autophagy marker protein ATG8-II in whitefly midguts shows that autophagy plays a critical role in repressing the infection of TYLCV in the whitefly27.
Here, using TYLCV as an example, we describe a protocol for the localization of begomoviruses in whitefly midguts, PSGs, and ovaries by an immunofluorescence technique. The technique includes dissection, fixation, and incubation with primary and dye-labeled secondary antibodies. Fluorescence signals showing the location of viral proteins in the whitefly tissues can then be detected under a confocal microscope. More importantly, this protocol can be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV using SYBR Green-based qPCR in whitefly midguts, PSGs, hemolymph, and ovaries, that can be used to compare the amount of virus in different whitefly tissue samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>MultiSciences</td>
			<td>F0001</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-ph&#233;nylindole (DAPI)</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>MultiSciences</td>
			<td>A3828</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System</td>
			<td>Bio-RAD</td>
			<td>185-5201</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 549-goat anti-mouse</td>
			<td>Earthox</td>
			<td>E032310-02</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Monoclonal antibody (MAb 1C4)</td>
			<td></td>
			<td></td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sangon Biotech</td>
			<td>B548119-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>TB green premix Ex Taq (Tli RNase H Plus)</td>
			<td>TaKaRa</td>
			<td>RR820A</td>
			<td>qPCR master mix</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Thermofisher</td>
			<td>A41182</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissuelyzer</td>
			<td>Shaghai jingxin</td>
			<td>Tissuelyser-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>BBI life sciences</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BBI life sciences</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization and quantification of begomoviruses in whitefly tissues by immunofluorescence and quantitative pcr

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

The MEAM1 whiteflies of the B. tabaci complex and TYLCV were used as an example here to describe the procedures. The overview of the immunofluorescence and viral quantification procedures described in this manuscript is shown in Figure 1. Figure 2 shows representative results of immunofluorescence detection of TYLCV and DAPI staining in PSGs, midguts, and ovaries, indicating that TYLCV accumulated more in the PSGs and midguts, and less in the ovaries. <...

Watch this Scientific Journal Video about Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR at JoVE.com

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

localization-quantification-begomoviruses-whitefly-tissues

Research

JoVE Journal

Immunology and Infection

5.5K Views.  Zhejiang University. We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Video: Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...