<ol style="list-style-type:upper-alpha;">
<li>Preparation of phantom lesion (prepare phantom about 38 to 48 hours prior to actual practicing session of FNA proficiency (Figure 1) or utilize ready to use 'AV Phantom' from FNA Source, USA. The phantom lesion (stored under refrigeration without freezing) may be kept prepared even 3 to 4 days prior to practice session.</li>
<li>Practicing FNA proficiency: Although we have demonstrated the procedure using Shidham's THFV (Tissue Harvester with Functional Valve) (FNA Source, USA), any other FNA system may be used for practicing (Figure 2).</li>
<li>Spreading and processing the aspirate on the glass slides (Figure 3).</li>
</ol>
Material required
<ol style="list-style-type:lower-alpha;">
<li>Clear silicone rubber caulk- available at many construction/home improvement stores. Many brands are available including GETM, DAP, Red Devil, White Lightning, and local chain brands. We used Clear Silicone Rubber, White Lightning Products, Cleveland, Ohio, USA, <a href="http://www.wlcaulk.com/products/all_purpose/silicone_rubber/wl09010.html">http://www.wlcaulk.com/products/all_purpose/silicone_rubber/wl09010.html</a>)</li>
<li>Caulking gun</li>
<li>A piece of plastic wrap sheet (we used Saran Cling Plus Wrap, SC Johnson &amp; Son Inc, Racine, WI, USA <a href="http://www.saranbrands.com/plastic-wrap/index.asp">http://www.saranbrands.com/plastic-wrap/index.asp</a></li>
<li>Latex gloves</li>
<li>Wide diameter banana or sausage.</li>
<li>Firm and flat base plate (approximately 6 inches X 6 inches) made of thick card board, acrylic plate, or other.</li>
<li>FNA performing equipments 
<ul>
<li>25 gauge hypodermic needles</li>
<li>Shidham's THFV (or other components for performing FNA)</li>
<li>glass slides</li>
<li>staining kits</li>
</ul>
</li>
</ol>
Step-by-step details
A. Preparation of phantom lesion (prepare phantom 1 to 2 days prior to practicing FNA proficiency (Figure 2) or utilize ready to use 'AV Phantom' from FNA Source, USA.
<ol>
<li>Cut a 6 X 6 inch size of supporting board.</li>
<li>Cut a 2.5 inch long segment of 'phantom lesion core' (banana or sausage).</li>
<li>Expel 0.5 inch thick layer of caulk on the base plate.</li>
<li>Press the 'phantom lesion core' lightly in to this layer of caulk. </li>
<li>Add more caulk over the 'phantom lesion core' so that it is completely embedded in the mound of caulk, without trapping air bubbles.</li>
<li>Adjust and shape the caulk over 'phantom lesion core' with gloved hand.</li>
<li>Take a 6 X 6 inch size piece of plastic wrap and cover the caulk mound and adjust the surface of the mound into a smooth convex configuration with a clean, gloved hand.</li>
<li>Let the caulk mound cure for 6-8 hours longer than the curing time mentioned under the product instructions, (usually for 24 hours), in warm place.</li>
<li>After curing, the caulk should be firm, and the plastic wrap can be peeled off easily.</li>
<li>The FNA phantom lesion is ready to be used for practicing the FNA procedure after about 8 hours when the entire mound of caulk has completely cured &nbsp;to the center containing the 'phantom lesion core' (banana or sausage).</li>
</ol>
B. Practicing FNA proficiency on AV Phantom lesion.
Any approach in obtaining high quality, adequate FNA aspirates with good quality direct cytology smears should address following critical points-
<ol style="list-style-type:upper-roman;">
<li>The needle gauge- as implied in the title of the procedure, the finer needles (higher gauge number) yield better aspirates with minimal blood dilution. For critical balance of benefits and limitations, 22 to 25 gauge needles are preferred for optimal mechanical stability of the needle without affecting the FNA aspirate quality.</li>
<li>Controlling appropriate sequences in vacuum application and release during the FNA procedure- This is critical in order to advance the sampled cells with each stroke of the needle, towards the needle hub. However, the aspirated material should not be allowed to reach into the area of the needle aspiration assembly with syringe. If this is not prevented, the material may be difficult to retrieve for making smears (this material may, however, be used for cell block preparation or for concentration methods such as Cytospin, filter methods, or recent liquid based cytology preparations).</li>
<li>Developing appropriate skill in processing of the aspirated specimen, including direct smear preparation and on-site adequacy evaluation with appropriate triaging for ancillary tests such as cell-block preparation for elective immunocytochemistry, elective flow cytometry for immunophenotyping of lymphoproliferative lesions, submitting sterile passes for microbiology studies, and material for molecular tests (cytogenetics, etc.).</li>
</ol>
Ba. Performing FNA procedure (shown with Shidham's THFV [1] (FNA Source, USA); however, any other FNA system may be used for practicing) (Figure 3,4,5).
<ol>
<li>Prepare the FNA assembly with the valve closed. </li>
<li>Locate the lesion and insert the needle tip in the lesion.</li>
<li>Open the valve to connect the vacuum, through the needle to its tip, to aspirate representative cellular components of the lesion with each to and fro stroke into the lesion.</li>
<li>Maintain the vacuum and sample different areas of the lesion by inserting the needle into the lesion in different directions.</li>
<li>Close the valve to disconnect the vacuum in the needle lumen.</li>
<li>Equalize the pressure with ambient conditions (E.g. by disconnecting the Syringe-valve from the needle for a brief moment &amp; reconnect. If the special device with inbuilt steps, such as 'THFV' is used, this and other steps from Ba1 through Ba6 are concerted automatically by following different steps in the valve system) [1].</li>
<li>Remove the needle from the lesion with the valve closed, in order to prevent loss of the specimen by capillary action into the lesion unless there is slightly negative pressure at the hub end.</li>
<li>Dislodge the aspirated content onto the glass slide to prepare direct smear (see below) (figure 6).</li>
</ol>
Bb. Processing of the aspirated specimen with appropriate triage 
Spreading the aspirate on the glass slides and preparing good quality direct smears (Figure 6) followed by proper staining and triage (Figure 7,8).
<ol>
<li>A drop of aspirate dislodged on the glass slide is spread by a variety of approaches as shown in (Figure 6) [2]. The usual approach is 'a'. However, if the specimen is blood diluted with some microfragments, the excess blood is drained away by tilting the slide, and the microfragments then are spread between this slide and second glass slide similar to 'a'. If the aspirate is predominantly a suspension without microfragments, such as from lymphoproliferative lesions, it is spread similar to 'b' or 'c'.</li>
<li>The smears may be processed and fixed in variety of ways for different staining methods during on-site adequacy evaluation. However, the best approach is to air-dry all the smears. The air-dried smears can be stained with Romanowski stains (such as Difff-Quik stain, Wright stain) after methanol fixation or with Pap stain after saline-rehydration and post-fixation (in 95% ethanol or preferably in 95% ethanol with 5% acetic acid) [3]. Elimination of interference due to the obscuring blood is a significant benefit [3].</li>
<li>Depending on the initial cytomorphologic findings during on-site adequacy evaluation, additional triage (such as elective cell block, electron microscopy, microbiology cultures, flow cytometry, molecular tests-cytogenetics etc.) is recommended as indicated.</li>
</ol>
Representative Results:
<img border="0" src="/files/ftp_upload/1404/1404fig1tmb.jpg" alt="figure 1" /> <a href="https://www.jove.com/files/ftp_upload/1404/1404fig1.jpg">Please click here to see a larger version of figure 1</a>.
 
Figure 1. Material required for preparing AV Phantom for practicing FNA proficiency (*We use Saran plastic wrap which did not stick to the caulk at the end, while removing after complete curing of the caulk.)
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig2tmb.jpg" alt="figure 2" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig2.jpg">Please click here to see a larger version of figure 2.</a>
 
Figure 2. Preparing AV Phantom lesion
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig3tmb.jpg" alt="figure 3" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig3.jpg">Please click here to see a larger version of figure 3.</a>
 
Figure 3. Critical steps in FNA procedure.
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig4tmb.jpg" alt="figure 4" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig4.jpg">Please click here to see a larger version of figure 4.</a>
 
Figure 4. EUS-FNA- Summary of Critical steps* (see Fig 3)
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig5tmb.jpg" alt="figure 5" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig5.jpg">Please click here to see a larger version of figure 5.</a>
 
Figure 5. Pitfalls related to the critical steps in FNA procedure.
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig6tmb.jpg" alt="figure 6" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig6.jpg">Please click here to see a larger version of figure 6.</a>
 
Figure 6. Preparation of direct smears of FNA specimen between two slides.
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig7tmb.jpg" alt="figure 7" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig7.jpg">Please click here to see a larger version of figure 7.</a>
 
Figure 7. Wet fixed versus Air-dried smears.
<hr style="border-bottom:1px dotted #999999;" />

<img border="0" src="/files/ftp_upload/1404/1404fig8tmb.jpg" alt="figure 8" /> 
<a href="https://www.jove.com/files/ftp_upload/1404/1404fig8.jpg">Please click here to see a larger version of figure 8.</a>
 Figure 8.Wet fixed versus Air-dried smears.

<ol>
	<li>Shidham, V. B., Pandit, A. W., Rao, R. N., Basir, Z., Shidham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Harvester+with+Functional+Valve+(THFV):+Shidham's+device+for+reproducibly+higher+specimen+yield+by+fine+needle+aspiration+biopsy+with+easy+to+perform+steps.">Tissue Harvester with Functional Valve (THFV): Shidham's device for reproducibly higher specimen yield by fine needle aspiration biopsy with easy to perform steps.</a> BMC Clinical Pathology. 7 (2), (2007).</li><li>Shidham, V. B., Epple, J., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AppendiX+I:+Collection+and+processing+of+effusion+fluids+for+cytopathologic+evaluation.">AppendiX I: Collection and processing of effusion fluids for cytopathologic evaluation.</a> , 207-235 (2007).</li><li>Shidham, V., Kampalath, B., England, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+air+drying+of+all+the+smears+prepared+during+fine+needle+aspiration+and+intraoperative+cytology+studies:+An+opportunity+to+practice+a+unified+protocol,+offering+the+flexibility+of+choosing+variety+of+staining+methods.">Routine air drying of all the smears prepared during fine needle aspiration and intraoperative cytology studies: An opportunity to practice a unified protocol, offering the flexibility of choosing variety of staining methods.</a> Acta Cytologica. (45), 60-68 (2001).</li></ol>

Fine-needle aspiration biopsies (FNAB) are widely performed as an economical, safe, and rapid minimally invasive technique for tissue diagnosis of various tumors and lesions. In this procedure a 18 to 27 gauge needle is inserted into the lesion, moved back and forth multiple times in different areas of the lesion under controlled vacuum (suction) of variable intensity ranging from minimal in the form of only capillary action to about 10 ml vacuum in syringe to retrieve diagnostic tissue material. A finer gauge needle with higher number is preferred to retrieve good cytology material to prepare direct smears. Wider gauge needle of 18G retrieves more sample suitable for cell block, flow cytometry, electron microscopy, cytogenetics, microbiology cultures etc. Although the procedure is simple, controlling various steps with proper application and release of vacuum at appropriate steps in concerted manner is extremely crucial for optimum results. 
Due to non-availability of specially designed high-yield needles, conventional hypodermic needles or their modifications are used to perform this procedure. However, the hypodermic needles have many limitations leading to a low yield of specimen if used in conventional manner. The requirement of special syringe grips usually adds to the complexity of the procedure. Thus, due to the lack of commercially available suitable FNAB needle device, this highly versatile procedure suffers a frequent problem of inadequate specimen retrieval. Recently, a device increasing the yield with reproducibility has been reported [1].
In addition, currently there is a lack of suitable model for practicing proficiency in performing FNAB by health personnel in training including residents and fellows. We had standardized a model with a piece of fresh cattle liver stuffed in latex glove for practicing FNAB proficiency. However, this model is difficult to organize at a short notice. Procurement of fresh cattle liver is usually the limiting predicament. In addition, handling of liver may introduce contamination-related factors as a significant draw back. The tendency for the latex glove to leak after a few needle pricks leads to potential mess during the practice session.
We report and describe a novel but simple method of embedding a small piece of banana (or sausage) in a commercially available silicone rubber caulk and other easily available material. The FNAB phantom retains vacuum seal thus permitting aspiration of material from the embedded specimen, equivalent to the actual FNAB procedure on a lesion in clinical setting. In this video, we demonstrate the procedure of making FNAB phantom lesion and practicing FNAB procedure. It also demonstrates in brief the processing of retrieved specimen including smear preparation and staining [2,3]. 
Because the caulk takes time to cure, the phantom lesion should be prepared in advance about 24 to 48 hours prior to the practicing session. Comparable ready to use phantom lesion may be obtained from a commercial source (FNA Source, USA).
The phantom lesion for practicing FNAB proficiency may be used for training the residents and fellows prior to the beginning of FNAB performance on patients in the clinical setting.

preparation and using phantom lesions to practice fine needle aspiration biopsies

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) procedure. In the past, we standardized a model consisting of latex glove containing fresh cattle liver for practicing FNAB. However, this model is difficult to organize and prepare on short notice, with the procurement of fresh cattle liver being the most challenging aspect. Handling of liver with contamination-related problems is also a significant draw back. In addition, the glove material leaks after a few needle passes, with resulting mess.
We have established a novel simple method of embedding a small piece of sausage or banana in a commercially available silicone rubber caulk. This model allows the retention of vacuum seal and aspiration of material from the embedded specimen, resembling an actual FNAB procedure on clinical mass lesions.
The aspirated material in the needle hub can be processed similar to the specimens procured during an actual FNAB procedure, facilitating additional proficiency in smear preparation and staining.

Watch this Scientific Journal Video about Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies at JoVE.com

Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies

Practicing of fine needle aspiration biopsies (FNAB) by trainees is relatively challenging, due to the lack of an easily available, appropriate lesion. Preparation of an AV phantom lesion for practicing the FNAB procedure and mastering proficiency is relatively easy.

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) ...

preparation-using-phantom-lesions-to-practice-fine-needle-aspiration

Research

JoVE Journal

Biology

17.8K visualizações.  University of Wisconsin - Milwaukee. Prática de PAAF (PAAF) por estagiários é relativamente difícil, devido à falta de uma lesão facilmente disponível, apropriado. Preparação de uma lesão phantom AV para a prática do procedimento de PAAF e proficiência masterização é relativamente fácil.

Vídeo: Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This remodeling leads to increased pulmonary vascular resistance, worsened right ventricular function, and premature death. Currently approved therapies for PAH largely target pulmonary vasodilator pathways; however, recent emerging therapeutic modalities are focused on other novel pathways involved in the pathogenesis of the disease, including right ventricle (RV) remodeling. Imaging techniques that allow longitudinal assessment of novel therapeutics are very useful for determining the efficacy of new drugs in preclinical studies. Noninvasive trans-thoracic echocardiography remains the standard approach to evaluating heart function and is widely used in rodent models. However, echocardiographic evaluation of the RV can be challenging due to its anatomical position and structure. In addition, standardized guidelines are lacking for echocardiography&#160;in preclinical rodent models, making it difficult to carry out a uniform assessment of RV&#160;function across studies in different laboratories. In preclinical studies, the monocrotaline (MCT) injury model in rats is widely used to evaluate drug efficacy for treating PAH. This protocol describes the echocardiographic evaluation of the RV in na&#239;ve and MCT-induced PAH rats.

The present protocol describes the echocardiographic characterization of right ventricular morphology and function in a rat model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This ...

Using Skin Biopsies to Generate Patient-Derived Podocytes

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Author Spotlight: Generation of Patient-Derived Podocytes from Skin Biopsies  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...