Tissue Embedding Research Articles

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

A Method for Fixing and Paraffin Embedding Tissue to Retain the Natural Fluorescence of Reporter Proteins

Protocol Summary Here we present an ethanol fixation protocol that allows for the direct visualization of the natural fluorescence of reporter proteins while maintaining excellent tissue histology. We demonstrate the utility of this method for visualizing green and red fluorescent proteins in a wide range of murine tissues using both cytoplasmic and membrane-localized fluorescent reporter proteins. Tissues fixed by this method also allow for immunohistochemical studies, providing a single method to visualize the natural fluorescence of reporter proteins with subsequent detection of cellular proteins.

Precast Gelatin-Based Molds for Tissue Embedding Compatible with Mass Spectrometry Imaging.

Preparation of tissue for matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) generally involves embedding the tissue followed by freezing and cryosectioning, usually between 5 and 25 μm thick, depending on the tissue type and the analyte(s) of interest. The brain is approximately 60% fat; it therefore lacks rigidity and poses structural preservation challenges during sample preparation. Histological sample preparation procedures are generally transferable to MALDI-MSI; however, there are various limitations. Optimal cutting temperature compound (OCT) is commonly used to embed and mount fixed tissue onto the chuck inside the cryostat during cryosectioning. However, OCT contains potential interferences that are detrimental to MALDI-MSI, while fixation is undesirable for the analysis of some analytes either due to extraction or chemical modification (i.e., polar metabolites). Therefore, a method for both fixed and fresh tissue compatible with MALDI-MSI and histology is desirable to increase the breadth of analyte(s), maintain the topographies of the brain, and provide rigidity to the fragile tissue while eliminating background interference. The method we introduce uses precast gelatin-based molds in which a whole mouse brain is embedded, flash frozen, and cryosectioned in preparation for mass spectrometry imaging (MSI).

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Enhanced Histochemical Detection of Iron in Paraffin Sections of Mouse Central Nervous System Tissue: Application in the APP/PS1 Mouse Model of Alzheimer's Disease.

Histochemical methods of detecting iron in the rodent brain result mainly in the labeling of oligodendrocytes, but as all cells utilize iron, this observation suggests that much of the iron in the central nervous system goes undetected. Paraffin embedding of tissue is a standard procedure that is used to prepare sections for microscopic analysis. In the present study, we questioned whether we could modify the iron histochemical procedure to enable a greater detection of iron in paraffin sections. Indeed, various modifications led to the widespread labeling of iron in mouse brain tissue (for instance, labeling of neurons and neuropil). Sites of focal concentrations, such as cytoplasmic punctate or nucleolar staining, were also observed. The modified procedures were applied to paraffin sections of a mouse model (APP/PS1) of Alzheimer’s disease. Iron was revealed in the plaque core and rim. The plaque rim had a fibrillary or granular appearance, and it frequently contained iron-labeled cells. Further analysis indicated that the iron was tightly associated with the core of the plaque, but less so with the rim. In conclusion, modifications to the histochemical staining revealed new insights into the deposition of iron in the central nervous system. In theory, the approach should be transferrable to organs besides the brain and to other species, and the underlying principles should be incorporable into a variety of staining methods.

A toolkit of thread-based microfluidics, sensors, and electronics for 3D tissue embedding for medical diagnostics.

Threads, traditionally used in the apparel industry, have recently emerged as a promising material for the creation of tissue constructs and biomedical implants for organ replacement and repair. The wicking property and flexibility of threads also make them promising candidates for the creation of three-dimensional (3D) microfluidic circuits. In this paper, we report on thread-based microfluidic networks that interface intimately with biological tissues in three dimensions. We have also developed a suite of physical and chemical sensors integrated with microfluidic networks to monitor physiochemical tissue properties, all made from thread, for direct integration with tissues toward the realization of a thread-based diagnostic device (TDD) platform. The physical and chemical sensors are fabricated from nanomaterial-infused conductive threads and are connected to electronic circuitry using thread-based flexible interconnects for readout, signal conditioning, and wireless transmission. To demonstrate the suite of integrated sensors, we utilized TDD platforms to measure strain, as well as gastric and subcutaneous pH in vitro and in vivo.

Abstract 1377: Impact of biospecimen pre-analytical factors on molecular analysis

Abstract The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1377.

Influence of three different histological methods on the morphology and morphometrical data in human testis.

Coagulant fixatives and paraffin embedding were widely used in the past for histomorphometrical evaluations of the human testis under physiological and pathological conditions. However, new methods are applied nowadays using better combinations of fixatives and plastic resins as embedding media, improving cell and tissue structural preservation. In an attempt to compare old and new data, the present study evaluated histomorphometrical data obtained from human testis after three different histological processing methods: Bouin/paraplast, glutaraldehyde/glycol methacrylate and glutaraldehyde/araldite. The morphometrical parameters were not affected by glutaraldehyde fixation after both resin embedding (methacrylate or araldite). On the other hand, Bouin/paraplast embedding lead to tissue shrinkage, which could give rise to misinterpretations on the measurements performed. Since some germ and somatic cells recognition do not depend upon high resolution techniques, counting of such cell types could be performed even using routine Bouin/paraplast protocols. Thus, the morphometrical analyses relying on cell recognition were not affected by the methods here applied, however, when metric measurements were applied, the obtained results could not be promptly compared. However, if the study requires confident spermatogonial identification for kinetics evaluation, glutaraldehyde/araldite processing is highly recommended.

Resident Lung Macrophages Are Sparsely Amongst Alveoli

Lung macrophages are major determinants of pro‐ and anti‐inflammatory mechanisms underlying multiple lung diseases, including Acute Lung Injury (ALI). Our recent findings in optically imaged mouse lungs indicate that resident macrophages in alveoli adhere to the alveolar epithelium (Nature 2014). We called these cells sessile alveolar macrophages (SAMs) to distinguish them from non‐adherent macrophages that can be recovered in the bronchoalveolar lavage. Here we report new studies in which we determined distribution of SAMs by lung histology. We anesthetized mice, then exsanguinated them by cardiac puncture (n=3). Through a tracheal cannula we instilled tissue embedding compound (OCT, R&D Systems) diluted 1:1 in PBS to inflate the lungs at 25 cmH2O. After 20 min, we isolated the lungs, froze the lungs in dry ice, then stored the lungs overnight at −80°C. We obtained tissue blocks that we sectioned in a tissue microtome to obtain 8 micron sections at 100–200 microns below the pleural surface. For immunohistochemistry, we incubated the sections at 4°C overnight with primary antibodies against the lung macrophage markers, CD11c and Siglec‐F, and the primary antibody against the alveolar wall marker, connexin 43 (Cx43). Then after washing the sections, we further exposed them to secondary antibodies tagged with different fluorescent markers. We viewed the sections by fluorescence microscopy. We identified alveolar walls as Cx43 expressing cells (green), and SAMs as cells co‐expressing CD11c (cyan) and Siglec‐F (red). In each lung, we counted the number of SAMs in 100 alveoli in 7–8 imaging fields. Normalized for a single imaging field, our counts indicated that 5±2 SAMs were present in 14±5 alveoli, indicating that SAMs were distributed as approximately 1 per 3 alveoli. We conclude, SAMs are sparsely distributed in the parenchyma, suggesting that SAMs employ communicative strategies for pathogen detection in macrophage‐free alveoli (Support: HL57556).Support or Funding InformationSupport: HL57556

An Improved Immunostaining and Imaging Methodology to Determine Cell and Protein Distributions within the Bone Environment.

Bone is a dynamic tissue that undergoes multiple changes throughout its lifetime. Its maintenance requires a tight regulation between the cells embedded within the bone matrix, and an imbalance among these cells may lead to bone diseases such as osteoporosis. Identifying cell populations and their proteins within bone is necessary for understanding bone biology. Immunolabeling is one approach used to visualize proteins in tissues. Efficient immunolabeling of bone samples often requires decalcification, which may lead to changes in the structural morphology of the bone. Recently, methyl-methacrylate embedding of non-decalcified tissue followed by heat-induced antigen retrieval has been used to process bone sections for immunolabeling. However, this technique is applicable for bone slices below 50-µm thickness while fixed on slides. Additionally, enhancing epitope exposure for immunolabeling is still a challenge. Moreover, imaging bone cells within the bone environment using standard confocal microscopy is difficult. Here we demonstrate for the first time an improved methodology for immunolabeling non-decalcified bone using a testicular hyaluronidase enzyme-based antigen retrieval technique followed by two-photon fluorescence laser microscopy (TPLM) imaging. This procedure allowed us to image key intracellular proteins in bone cells while preserving the structural morphology of the cells and the bone.

An improved approach to align and embed multiple brain samples in a gelatin-based matrix for simultaneous histological processing

BackgroundPreparation and processing of free-floating histological sections involve a series of steps. The amount of labor, particularly sectioning and mounting, quickly multiplies as the number of samples increases. Embedding tissue samples in a flexible matrix allows simultaneous handling of multiple samples and preserves the integrity of the tissue during histological processing. However, aligning multiple asymmetrical samples, for example small-animal brains, in a particular orientation requires skillful arrangement and securing of the samples by pinning onto a solid surface. Consequently, costly technical services offered by contract research organizations are often sought. New methodAn improved approach to align and embed multiple whole or half rodent brain samples into a gelatin-based matrix is described. Using a template specifically designed to form arrayed mouse brain-shaped cavities, a ⿿receiving matrix⿿ is prepared. Inserting brain samples directly into the cavities allows the samples to be effortlessly positioned into a uniform orientation and embedded in a block of matrix. ResultsMultiple mouse brains were arrayed in a uniform orientation in a gelatin matrix block with ease using the receiving matrix. The gelatin-embedded brains were simultaneously sectioned and stained, and effortlessly mounted onto glass slides. Comparison with existing methodsThe improved approach allowed multiple whole or half mouse brains to be easily arrayed without pinning the samples onto a solid surface and prevented damages or shifting of the samples during embedding. ConclusionsThe new approach to array multiple brain samples provides a simple way to prepare gelatin-embedded whole or half brain arrays of commercial quality.

Inferior Vena Cava Filters: Placement and Retrieval

The incidence of venous thromboembolism, including both deep venous thrombosis and pulmonary embolism, is approximately 100 per 100,000 patients in the United States. Although anticoagulation is the mainstay of treatment, increasing numbers of patients with absolute contraindications, treatment failures, and complications has led to rising numbers of inferior vena cava (IVC) filter placements. IVC filter placements, however, are not without potential early and long-term complications such as filter thrombosis, perforation, migration, fracture, and embolization. Although IVC filter removal is successful in many cases, retrieval may be technically challenging, and ultimately unsuccessful, due to associated thrombus, substantial filter tilting, filter fracture, and excessive tissue embedding surrounding the tip or struts. As a result of increasing prevalence of IVC filters, it is important that all members of the health care team be comfortable with these devices. The purpose of this article is to provide a comprehensive review of IVC filters, their placement, and retrieval.

A method for fixing and paraffin embedding tissue to retain the natural fluorescence of reporter proteins.

Green fluorescent protein (GFP) and its derivatives are routinely employed as surrogate markers for gene expression and lineage tracing in genetically engineered mice. Tissues from these mice are commonly formalin fixed and paraffin embedded (FFPE) for histological studies. However, this results in inactivation of the natural fluorescence of these proteins, requiring their detection by immunological techniques. Here we present an ethanol fixation protocol that allows for the direct visualization of the natural fluorescence of reporter proteins while maintaining excellent tissue histology. We demonstrate the utility of this method for visualizing green and red fluorescent proteins in a wide range of murine tissues using both cytoplasmic and membrane-localized fluorescent reporter proteins. Tissues fixed by this method also allow for immunohistochemical studies, providing a single method to visualize the natural fluorescence of reporter proteins with subsequent detection of cellular proteins.

Expression and pathological mechanism of MMP-9 and HIF-2α in CD133(+) lung cancer stem cells

To investigate the expression and pathological mechanism of matrix metalloproteinase (MMP)-9 and hypoxia-inducible factor (HIF)-2α in CD133⁺ lung cancer stem cells. Sixty-two cases of lung cancer paraffin embedding tissues were collected from the First Affiliated Hospital of Soochow University between January 2009 and December 2009. Immunohistochemistry (IHC) was used for detection of CD133 expression in lung cancer tissues and the clinical significance was analyzed. Real-time polymerase chain reaction (PCR) was used for the investigation of expression of tumor metastasis associated genes, including MMP-1, MMP-2, MMP-9, HIF-1α, HIF-1β, HIF-2α and tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, TIMP-4. Scrambled siRNA or CD133 siRNA were used to transfect the lung cancer cell line A549, then Control-si-A549 cells and CD133-si-A549 cells were generated respectively. PCR was used to analysis CD133, MMP-9 and HIF-2α genes expression and transwell invasion assay was used to study the invasion ability of A549 cells in two groups. 51.6% of lung cancer tissues expressed CD133 (P<0.05); the expression level of CD133 was related to tumor metastasis and patients' survival rate (P<0.05). The gene expression of HIF-2α and MMP-9 were increased in CD133⁺ lung cancer cells compared with CD133⁻ cancer cells (1.58 ± 0.39 vs 1.10 ± 0.31, 1.67 ± 0.38 vs 1.05 ± 0.21, all P<0.05), whereas no difference was found in gene expression of MMP-1, MMP-2, HIF-1α, HIF-1β and TIMP-1, TIMP-2, TIMP-3, TIMP-4 (all P>0.05). Compared with the Control-si-A549 cell, the expression of CD133, HIF-2α and MMP-9 (0.24 ± 0.10 vs 0.85 ± 0.23, 0.19 ± 0.09 vs 0.54 ± 0.18, 0.31 ± 0.17 vs 1.12 ± 0.31, all P<0.05) in CD133-si-A549 cell were remarkably decreased. The number of CD133-si-A549 cells migrated to below room was significantly smaller than that of Control-si-A549 cells (207 ± 25 vs 82 ± 10, P<0.05). The CD133⁺ lung cancer stem cell is correlated to the tumor metastasis and patients' survival. CD133⁺ tumor stem cell can promote the tumor invasion and metastasis via the up-regulation of HIF-2α and MMP-9 expression.

El endometrio humano - Cambios histoquímicos y ultraestructurales durante el ciclo menstrual normal

El desarrollo de las técnicas de fijación, inclusión y corte de tejidos para microscopía electrónico, hizo posible que, desde comienzos de la década del 60, se acumulase información valiosa sobre la ultraestructura del endometrio de diversas especies animales y de la mujer en condiciones normales y de actividad hormonal. Los trabajos de Ancla y colaboradores, de Nilsson, de Clyman, Tarzakis y de Cavazos y colaboradores ponen en evidencia lo acotado en lo que a detalles ultraestructurales del endometrio de la mujer durante el ciclo menstrual normal se refiere.

How to submit a nail specimen.

The scarcity of specific submission protocols for nail unit biopsies presents many challenges for appropriate specimen processing. Many nail biopsies are received fragmented or without orientation, often resulting in less-than-ideal tissue embedding and poor histologic sections, which are difficult to interpret. Methods are described for proper nail matrix/bed biopsy and plate submission that incorporate aspects of previous submission protocols and include inking the biopsy specimen along with submitting the tissue on a drawing of the nail. Also described is a technique for maintaining adherence of nail plate to glass slides, a chronic challenge in the laboratory.

Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory

Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more) either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 μ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

Abstract 3497: A novel method of active paraffin removal and efficient extraction of NGS-quality DNA from FFPE tissues

Abstract Formalin Fixation and Paraffin Embedding (FFPE) of tissues remains a routine histology practice by pathologists due to the quality and stability of structural information preserved. The method was conceived primarily to facilitate histological analysis rather than molecular approaches increasingly common in clinical oncology settings such as targeted sequencing and whole genome sequencing. Chemical crosslinks and the paraffin matrix of FFPE-preserved samples present significant challenges for the robust extraction of nucleic acids. To address this and provide reliable access to clinical samples, we present a novel method for consistent and reproducible extraction and purification of DNA from FFPE samples using Covaris Adaptive Focused Acoustics (AFA). Use of focused ultrasonic energy to emulsify embedding paraffin. The method is predicated on the use of highly controlled acoustic energy to efficiently remove paraffin from FFPE cores, sections, and slides enabling efficient tissue rehydration, tissue digestion, crosslink reversal, and nucleic acid release. In addition, this critical step is carried out without the use of organic solvents or mineral oils. We present quantitative fluorescent microscopy illustrating the efficiency of paraffin removal from tissues utilizing this novel approach. Better data quality as a consequence of improved DNA yield and quality. Having established reproducible sample processing we sought to explore benefits in downstream applications and analyses. Results, utilizing replicates of several different tissue types, indicate high dsDNA yields, controllable fragment sizes and excellent DNA quality as indicated by commercial qPCR based kits designed to assess DNA quality. Improved sequencing coverage as a consequence of improved DNA yield and quality. The quality of the extracted DNA, as a function of consistent sequence coverage across the entire genome, was then confirmed using whole genome sequencing of the FFPE samples as compared to sequence data obtained from matched fresh frozen tissue. Our data also indicate that the high quality controllable DNA extraction allows for preparation of large insert (1-6kb) libraries previously considered very difficult from FFPE stabilized tissues. Method suitable for automation. The simple workflow allows for the parallel processing of up to 8 FFPE samples in a 96 well plate format. We present data showing highly reproducible results while processing a large number of FFPE samples. Citation Format: Hamid Khoja, Edwin Rudd, Austin Purdy, James Han, Srikanth Kakumanu, Adrian Palmer, Guillaume Durin, Jim Laugharn. A novel method of active paraffin removal and efficient extraction of NGS-quality DNA from FFPE tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2014-3497

Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue.

BackgroundAn important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment. In situ PCR is a relatively simple and rapid method that involves the amplification of specific mRNA directly within plant tissue whilst incorporating labelled nucleotides that are subsequently detected by immunohistochemistry. Another notable advantage of this technique is that it can be used on plants that are not easily genetically transformed.ResultsAn optimised workflow for in-tube and on-slide in situ PCR is presented that has been evaluated using multiple plant species and tissue types. The protocol includes optimised methods for: (i) fixing, embedding, and sectioning of plant tissue; (ii) DNase treatment; (iii) in situ RT-PCR with the incorporation of DIG-labelled nucleotides; (iv) signal detection using colourimetric alkaline phosphatase substrates; and (v) mounting and microscopy. We also provide advice on troubleshooting and the limitations of using fluorescence as an alternative detection method. Using our protocol, reliable results can be obtained within two days from harvesting plant material. This method requires limited specialized equipment and can be adopted by any laboratory with a vibratome (vibrating blade microtome), a standard thermocycler, and a microscope. We show that the technique can be used to localise gene expression with cell-specific resolution.ConclusionsThe in situ PCR method presented here is highly sensitive and specific. It reliably identifies the cellular expression pattern of even highly homologous and low abundance transcripts within target tissues, and can be completed within two days of harvesting tissue. As such, it has considerable advantages over other methods, especially in terms of time and cost. We recommend its adoption as the standard laboratory technique of choice for demonstrating the cellular expression pattern of a gene of interest.

Large-scale screening of rice accessions to evaluate resistance to bakanae disease

Bakanae disease is an important fungal disease in the world. No rice (Oryza sativa L.) varieties have been found to be completely resistant to this disease. To facilitate accurate, uniform and simultaneous screening of many rice accessions, we developed an inoculation method for microconidia of Fusarium fujikuroi using a tissue embedding cassette and seedling tray. Standards for evaluating the inoculated rice seedlings as healthy or unhealthy were also established. The method was fast and reproducible for accurately evaluating resistance to bakanae disease in rice.