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Journal of Visualized Experiments : JoVE Mar 2018Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of...
Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 µm in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.
Topics: Animals; Female; Humans; Kidney; Kidney Transplantation; Male; Mice; Microdissection; Tissue Engineering
PubMed: 29658916
DOI: 10.3791/57358 -
Journal of Visualized Experiments : JoVE Apr 2020Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular...
Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.
Topics: Animals; Brain Neoplasms; Glioma; Humans; Laser Capture Microdissection; Mice; Neoplasm Invasiveness; Sequence Analysis, RNA; Staining and Labeling
PubMed: 32338655
DOI: 10.3791/60939 -
Annals of the New York Academy of... Nov 2020The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo... (Review)
Review
The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo models, progress to elucidate mechanisms of toxicity and to develop effective therapies, decontamination procedures, and general countermeasures is still limited. Novel scientific approaches to address these questions are needed to expand perspectives on existing knowledge and gain new insights. To achieve this, the use of ex vivo techniques like precision-cut tissue slices (PCTSs) can be a valuable approach. Existing studies employing this economical and relatively easy to implement method show model suitability and comparability with the use of in vitro and in vivo models. In this article, we review research on CWAs in PCTSs to illustrate the advantages of the approach and to promote future applications.
Topics: Animals; Chemical Warfare Agents; Humans; Microdissection
PubMed: 32808309
DOI: 10.1111/nyas.14459 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jan 2019Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and... (Review)
Review
Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.
Topics: Animals; Cell Separation; DNA; Laser Capture Microdissection; RNA; Single-Cell Analysis
PubMed: 30756532
DOI: 10.13345/j.cjb.180102 -
Journal of Visualized Experiments : JoVE Mar 2022Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic "toolkits,"...
Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic "toolkits," such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis. LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species. Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.
Topics: Animals; Laser Capture Microdissection; Lasers; Male; Proteins; Transcriptome
PubMed: 35435919
DOI: 10.3791/63666 -
Methods in Molecular Biology (Clifton,... 2012This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks,...
This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks, cryostat sectioning, processing slides, performing the LCM, and purification of RNA from LCM samples. Notes describe the fine points of each operation, which can often mean the difference between success and failure.
Topics: Animals; Cryoultramicrotomy; Equipment Design; Kidney; Laser Capture Microdissection; RNA
PubMed: 22639264
DOI: 10.1007/978-1-61779-851-1_19 -
Anatomical Record (Hoboken, N.J. : 2007) Nov 2013
Topics: Animals; Genomics; Humans; Laser Capture Microdissection; Pathology, Molecular; Proteomics
PubMed: 24123978
DOI: 10.1002/ar.22791 -
Molecular & Cellular Proteomics : MCP Dec 2022Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from...
Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug-target interactions using existing protein/drug binding databases.
Topics: Islets of Langerhans; Proteomics; Proteins; Laser Capture Microdissection
PubMed: 36244662
DOI: 10.1016/j.mcpro.2022.100426 -
Cytometry. Part a : the Journal of the... Dec 2019Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable...
Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Topics: Animals; Biological Specimen Banks; Chromosomes; DNA Probes; Humans; In Situ Hybridization, Fluorescence; Microdissection
PubMed: 31532073
DOI: 10.1002/cyto.a.23896 -
Lab on a Chip Jul 2022Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most...
Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample amounts that blurs cell-type-specific or microstructure-specific information. In this study, we developed an improved sample preparation approach for spatial proteomics and integrated it with our previously-established laser capture microdissection (LCM) and microfluidics sample processing platform. Specifically, we developed a hanging drop (HD) method to improve the sample recovery by positioning a nanowell chip upside-down during protein extraction and tryptic digestion steps. Compared with the commonly-used sitting-drop method, the HD method keeps the tissue pixel away from the container surface, and thus improves the accessibility of the extraction/digestion buffer to the tissue sample. The HD method can increase the MS signal by 7 fold, leading to a 66% increase in the number of identified proteins. An average of 721, 1489, and 2521 proteins can be quantitatively profiled from laser-dissected 10 μm-thick mouse liver tissue pixels with areas of 0.0025, 0.01, and 0.04 mm, respectively. The improved system was further validated in the study of cell-type-specific proteomes of mouse uterine tissues.
Topics: Animals; Laser Capture Microdissection; Mice; Proteome; Proteomics; Specimen Handling; Workflow
PubMed: 35838077
DOI: 10.1039/d2lc00384h