Current protocols最新文献

{"title":"Genetically Engineered and Implantable Mouse Brain Tumor Models: Characterization by Immunohistochemistry and Flow Cytometry","authors":"Apoorva Mirji,&nbsp;Gurveer Singh,&nbsp;Anzar A. Mujeeb,&nbsp;Brandon L. McClellan,&nbsp;YingXiang Li,&nbsp;Makayla Perez,&nbsp;Maria G. Castro","doi":"10.1002/cpz1.70080","DOIUrl":"10.1002/cpz1.70080","url":null,"abstract":"<p>Gliomas are aggressive tumors with a poor prognosis. The protocols presented here outline the methods used to study tumor progression, the tumor microenvironment (TME), and the effects of experimental treatments. The Sleeping Beauty (SB) transposase system induces tumors <i>de novo</i> to generate mouse models that recapitulate human gliomas. Plasmids are constructed with oncogenic drivers and other genetic alterations of interest. which are recognized by their unique position in between inverted/direct repeat (IR/DR) sequences. Luciferase enzyme is used to monitor the uptake of the plasmid, tumor growth, and response to experimental therapies. The genes of interest are tracked using fluorescent markers. Tumors will arise in immunocompetent hosts, which provides a relevant preclinical platform for analysis of tumor initiation, progression, survival, immune microenvironment, and histopathological features. Once the tumor grows within the desired brain location, it can be harvested to generate cell cultures of neurospheres for future experimentation. The benefit of implantable models generated from SB tumors is that they provide specific anatomical and genetic context, in which specific genetic characteristics can be tracked, as they are co-expressed with fluorescent markers. Post glioma cell implantation, additional analysis of the TME and tumor growth can be performed through immunohistochemistry (IHC) and flow cytometry. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Creation of mouse glioma models by Sleeping-Beauty-mediated transposition</p><p><b>Basic Protocol 2</b>: Generation of orthotopic implantable brain tumors and neurospheres</p><p><b>Basic Protocol 3</b>: Hematoxylin and eosin staining of glioma tissue samples</p><p><b>Basic Protocol 4</b>: Immunohistochemistry of glioma tissue samples</p><p><b>Basic Protocol 5</b>: Flow cytometry for immune cell analysis of the tumor microenvironment</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeted C-to-T Base Editing in the Arabidopsis Plastid Genome","authors":"Issei Nakazato,&nbsp;Shin-ichi Arimura","doi":"10.1002/cpz1.70075","DOIUrl":"10.1002/cpz1.70075","url":null,"abstract":"<p><i>Arabidopsis thaliana</i>, particularly the ecotype Columbia-0 (Col-0), has been extensively employed in the study of genetics of the nuclear genome. However, the difficulty of modifying the plastid genome of Col-0, the most widely used ecotype, has hindered investigation of the functional interactions between nuclear-encoded and plastid-encoded genes in this ecotype. Recently, we achieved targeted base editing, substituting a specific C:G pair with a T:A pair in the plastid genome of Col-0 through the application of genome-editing technology. This article introduces the method employed to accomplish this targeted base editing. The process involves four steps: (i) designing and constructing a binary vector encoding the genome-editing enzyme, (ii) introducing the binary vector into the nuclear genome of Col-0 via floral dipping, (iii) identifying base-edited plants, and (iv) verifying inheritance of the edited base(s) by the next generation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Design and construction of a binary vector encoding ptpTALECD or ptpTALECD_v2</p><p><b>Basic Protocol 2</b>: <i>Agrobacterium</i>-mediated introduction of a binary vector into the <i>Arabidopsis</i> nuclear genome</p><p><b>Basic Protocol 3</b>: Selection of plants harboring T-DNA in the nucleus and detection of base editing in the plastid genome</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70075","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142934111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simple Isolation of Human Bone Marrow Adipose Tissue–Derived Mesenchymal Stem/Stromal Cells","authors":"Gülsena Tonyalı,&nbsp;Emine Kiliç,&nbsp;Bihter Muratoğlu,&nbsp;Esin Alpdündar-Bulut,&nbsp;Cansu Özdemir,&nbsp;Duygu Uçkan-Çetinkaya","doi":"10.1002/cpz1.70081","DOIUrl":"10.1002/cpz1.70081","url":null,"abstract":"<p>Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell &amp; Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of human BMAT-MSCs from the fatty layer of the bone marrow</p><p><b>Basic Protocol 2</b>: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCs</p><p><b>Support Protocol 1</b>: Immunophenoypic characterization of human BMAT-MSCs by flow cytometry</p><p><b>Support Protocol 2</b>: In vitro characterization of multilineage differentiation potential of human BMAT-MSCs</p><p><b>Support Protocol 3</b>: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142924299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Searching and Using MobiDB Resource 6 to Explore Predictions and Annotations for Intrinsically Disordered Proteins","authors":"Maria Cristina Aspromonte,&nbsp;Federica Quaglia,&nbsp;Alexander Miguel Monzon,&nbsp;Damiano Clementel,&nbsp;Alessio Del Conte,&nbsp;Damiano Piovesan,&nbsp;Silvio C. E. Tosatto","doi":"10.1002/cpz1.70077","DOIUrl":"10.1002/cpz1.70077","url":null,"abstract":"<p>Intrinsically disordered proteins (IDPs) make up around 30% of eukaryotic proteomes and play a crucial role in cellular processes and in pathological conditions such as neurodegenerative disorders and cancers. However, IDPs exhibit dynamic conformational ensembles and are often involved in the formation of biomolecular condensates. Understanding the function of IDPs is critical to research in many areas of science. MobiDB is a unique resource that serves as a comprehensive knowledgebase of IDPs and intrinsically disordered regions (IDRs), combining disorder annotations from experimental evidence and predictions for a broad range of protein sequences. Over the past decade, MobiDB has evolved with a focus on expanding annotation coverage, standardizing annotation provenance, and enhancing database accessibility. The latest MobiDB, version 6, released in July 2024, includes significant improvements, such as the integration of AlphaFoldDB predictions and a new homology transfer pipeline that has substantially increased the number of entries with high-quality annotations. The user interface has also been updated, highlighting annotation features, clarifying the entry page, and providing an immediate overview of disorder, binding, and disorder functions information in the protein sequence. This protocol guides the user through applications of the MobiDB, including disorder prediction, curated data analysis, and exploration of interaction data. This guide covers how to perform a search in MobiDB annotations using the web interface and the MobiDB REST API for programmatic access. The protocols use a step-by-step walkthrough using the human growth hormone receptor to demonstrate MobiDB's functions for visualization and interpretation of protein disorder data. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Searching MobiDB query formats</p><p><b>Basic Protocol 2</b>: Searching MobiDB selected datasets and selected proteomes</p><p><b>Basic Protocol 3</b>: Performing a search on the Statistics page in MobiDB</p><p><b>Support Protocol</b>: Programmatic access with MobiDB REST API</p><p><b>Basic Protocol 4</b>: Visualizing and interpreting a MobiDB Entry: The GHR use case</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11668231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using Vesicular Stomatitis Virus as a Platform for Directed Protease Evolution","authors":"Francesco Costacurta,&nbsp;Stefanie Rauch,&nbsp;Dorothee von Laer,&nbsp;Emmanuel Heilmann","doi":"10.1002/cpz1.70074","DOIUrl":"10.1002/cpz1.70074","url":null,"abstract":"<p>Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain-of-function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS-CoV-2, MERS-CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Using VSV as a platform for directed protease evolution</p><p><b>Alternate Protocol</b>: Dose response assay with TCID₅₀ readout</p><p><b>Support Protocol 1</b>: A pipeline for high-throughput VSV sequencing</p><p><b>Support Protocol 2</b>: Rescue of VSV</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11664493/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Employment of a Newly Defined In Vitro Fertilization Protocol to Determine the Cytoskeletal Machinery, DNA Damage, and Subsequent DNA Repair Resulting from Endocrine Disruption by Hexavalent Chromium in Rat Metaphase II Oocytes","authors":"Liga Wuri,&nbsp;Paul W. Zarutskie,&nbsp;Joe A. Arosh,&nbsp;Sakhila K. Banu","doi":"10.1002/cpz1.70060","DOIUrl":"10.1002/cpz1.70060","url":null,"abstract":"<p>These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing <i>in vitro</i> fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: <i>In vivo</i> EDC treatment of rats and immunostaining of treated oocytes</p><p><b>Basic Protocol 2</b>: <i>In vitro</i> fertilization and immunostaining of early-stage embryos</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879207","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation, Purification, and Comprehensive Flow Cytometry Assessment of Lung Stromal Cells","authors":"Sophia Rottmann,&nbsp;Veronika Lukacs-Kornek","doi":"10.1002/cpz1.70078","DOIUrl":"10.1002/cpz1.70078","url":null,"abstract":"<p>Stromal cells are non-hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID-19 or flu infection.</p><p>To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex-vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of stromal cells from murine lung tissue</p><p><b>Basic Protocol 2</b>: Flow cytometry assessment of lung stromal populations</p><p><b>Basic Protocol 3</b>: Purification of lung fibroblastic stromal cells</p><p><b>Alternate Protocol</b>: Positive selection of fibroblastic stromal cells</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70078","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Microphysiological Cartilage-on-Chip Platform for Dynamic Biomechanical Stimulation of Three-Dimensional Encapsulated Chondrocytes in Agarose Hydrogels","authors":"Valtteri Peitso,&nbsp;Zahra Sarmadian,&nbsp;João Henriques,&nbsp;Elsa Lauwers,&nbsp;Carlo Alberto Paggi,&nbsp;Ali Mobasheri","doi":"10.1002/cpz1.70079","DOIUrl":"10.1002/cpz1.70079","url":null,"abstract":"<p>Osteoarthritis (OA) is one of the most prevalent joint diseases globally, characterized by the progressive breakdown of articular cartilage, resulting in chronic pain, stiffness, and loss of joint function. Despite its significant socioeconomic impact, therapeutic options remain limited, largely due to an incomplete understanding of the molecular mechanisms driving cartilage degradation and OA pathogenesis. Recent advances in <i>in vitro</i> modeling have revolutionized joint tissue research, transitioning from simplistic two-dimensional cell cultures to sophisticated three-dimensional (3D) constructs that more accurately mimic the physiological microenvironment of native cartilage. Over the last decade, organ-on-chip technologies have emerged as transformative tools in tissue engineering, offering microphysiological platforms with precise control over biomechanical and biochemical stimuli. These platforms are providing novel insights into tissue responses and disease progression and are increasingly integrated into the early stages of drug screening and development. In this article, we present a detailed experimental protocol for constructing a cartilage-on-chip system capable of delivering controlled dynamic biomechanical stimulation to 3D-encapsulated chondrocytes in an agarose hydrogel matrix. Our protocol, optimized for both bovine and human chondrocytes, begins with Basic Protocol 1, detailing the preparation and injection of cell-laden hydrogels into the microdevice. Basic Protocol 2 describes the application of dynamic mechanical loading using a calibrated pressurized pump. Finally, Basic Protocols 3 and 4 focus on the retrieval of the hydrogel and RNA extraction for downstream molecular analyses. This platform represents a critical advancement for <i>in vitro</i> studies of cartilage biology, enabling more precise modeling of OA pathophysiology and evaluation of experimental therapeutics. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Cartilage-on-chip injection</p><p><b>Basic Protocol 2</b>: Cartilage-on-chip actuation</p><p><b>Basic Protocol 3</b>: Cartilage-on-chip agarose hydrogel removal</p><p><b>Basic Protocol 4</b>: Preparation of cartilage-on-chip for RNA extraction</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Comprehensive Stereology Method for Quantitative Evaluation of Neuronal Injury, Neurodegeneration, and Neurogenesis in Brain Disorders","authors":"Doodipala Samba Reddy,&nbsp;Neo Zhu,&nbsp;Trisha Challa,&nbsp;Sai Gajjela,&nbsp;Hetvi Desai,&nbsp;Sreevidhya Ramakrishnan,&nbsp;Xin Wu","doi":"10.1002/cpz1.70053","DOIUrl":"10.1002/cpz1.70053","url":null,"abstract":"&lt;p&gt;Neuronal injury, neurodegeneration, and neuroanatomical changes are key pathological features of various neurological disorders, including epilepsy, stroke, traumatic brain injury, Parkinson's disease, autism, and Alzheimer's disease. Accurate quantification of neurons and interneurons in different brain regions is critical for understanding the progression of neurodegenerative disorders in animal models. Traditional scoring methods are often superficial, biased, and unreliable for evaluating neuropathology. Stereology, a quantitative tool that uses 3-dimensional visualization of cells, provides a robust protocol for evaluating neuronal injury and neurodegeneration. This article presents a comprehensive and optimized stereology protocol for unbiased quantification of neuronal injury, neurodegeneration, and neurogenesis in rat and mouse models. This protocol involves precise counting of injured neurons, surviving neurons, and interneurons through immunohistochemical processing of brain sections for NeuN(+) principal neurons, parvalbumin (PV+) interneurons, doublecortin (DCX+) newborn neurons, and Fluoro-Jade B (FJB+)-stained injured cells. Predefined hippocampal and amygdala regions were identified and analyzed using a Visiopharm stereology software-driven compound microscope. Cell density and absolute cell numbers were determined using the optical fractionation method. Our stereology protocol accurately estimated 1.5 million total NeuN(+) principal neurons and 0.05 million PV(+) interneurons in the rat hippocampus, as well as 1.2 million total principal neurons and 0.025 million interneurons in the mouse hippocampus. FJB(+) counting provided a quantitative index of damaged neurons, and the stereology of DCX(+) neurons demonstrated the extent of neurogenesis. Overall, this stereology protocol enables precise, accurate, and unbiased counting of total neurons in any brain region. This offers a reliable quantitative tool for studying neuronal injury and protection in various models of acute brain injury, neurotoxicity, and chronic neurological disorders. © 2024 Wiley Periodicals LLC.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 1&lt;/b&gt;: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in rat brain sections&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 2&lt;/b&gt;: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in mouse brain sections&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 3&lt;/b&gt;: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala in rats&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 4&lt;/b&gt;: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala regions in mice&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 5&lt;/b&gt;: Brain fixation and histology processing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 6&lt;/b&gt;: Immunochemistry of principal neurons, interneurons, and newborn neurons&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 7&lt;/b&gt;: Fluoro-Jade B staining of injured","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biotin-Based Northern Blotting (BiNoB): A Cost-Efficient Alternative for Detection of Small RNAs","authors":"Deeksha Madhry,&nbsp;Riya Roy,&nbsp;Bhupendra Verma","doi":"10.1002/cpz1.70065","DOIUrl":"10.1002/cpz1.70065","url":null,"abstract":"<p>Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin-based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity-based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3′-end labeled probes that were labeled in-house and 5′-end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3′-end labeled probes. Furthermore, we demonstrated that the use of an in-house buffer offered comparable sensitivity to a commercially available buffer, providing another cost-effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non-coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5-10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Biotin-based northern blotting</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}