Bio-protocol最新文献_第10页

Egg Microinjection for the Ladybird Beetle Harmonia axyridis. 瓢虫虫卵显微注射研究。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5136

Taro Nakamura, Yuji Matsuoka, Teruyuki Niimi

{"title":"Egg Microinjection for the Ladybird Beetle Harmonia axyridis.","authors":"Taro Nakamura, Yuji Matsuoka, Teruyuki Niimi","doi":"10.21769/BioProtoc.5136","DOIUrl":"10.21769/BioProtoc.5136","url":null,"abstract":"In this paper, we present a detailed protocol for microinjecting DNA, RNA, or protein solutions into fertilized eggs of the multicolored Asian ladybird beetle, Harmonia axyridis, under a stereomicroscope equipped with an injection apparatus. H. axyridis is an emerging model organism for studying various biological fields, showing intraspecific polymorphisms exhibiting highly diverse color patterns on the elytra. Here, we describe how to rear ladybird beetles in a laboratory and obtain fertilized eggs for microinjection experiments. We also provide a constant fluid flow injection method, which enhances the efficiency of microinjection and improves throughput. Our step-by-step protocol is applicable to generating transgenic or genome-edited ladybird beetles, facilitating functional genetics in H. axyridis; the microinjection method should be applicable to other insect eggs. Key features • Egg microinjection for the multicolored Asian ladybird beetle Harmonia axyridis under a stereomicroscope with injection equipment. • Detailed procedures for post-injection care, including rearing Harmonia axyridis. • Step-by-step guide for the efficient collection of Harmonia axyridis eggs.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5136"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904292","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}

引用次数: 0

Total Internal Reflection Fluorescence (TIRF) Single-Molecule Assay to Analyze the Motility of Kinesin. 全内反射荧光（TIRF）单分子法分析运动蛋白的运动性。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5135

Tomoki Kita, Shinsuke Niwa

引用次数: 0

An Autocatalytic Platform Combining a Nonlinear Hybridization Chain Reaction and DNAzyme to Detect microRNA. 结合非线性杂交链反应和DNAzyme的自催化平台检测微rna。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5134

Hongbo Zhang, Xiuen Cao, Qubo Zhu

{"title":"An Autocatalytic Platform Combining a Nonlinear Hybridization Chain Reaction and DNAzyme to Detect microRNA.","authors":"Hongbo Zhang, Xiuen Cao, Qubo Zhu","doi":"10.21769/BioProtoc.5134","DOIUrl":"10.21769/BioProtoc.5134","url":null,"abstract":"MicroRNAs (miRNAs) are small, non-coding RNAs that play pivotal roles in gene regulation; they are increasingly recognized as vital biomarkers for various diseases, notably cancer. Conventional methods for miRNA detection, such as quantitative PCR and microarray analysis, often entail intricate sample preparation and lack the requisite sensitivity to detect low-abundance miRNAs like miRNA-21. This protocol presents an innovative approach that combines branched hybridization chain reaction (bHCR) with DNAzyme technology for the precise detection of miRNA-21. The bHCR amplifies the target signal through a branched structure, while the DNAzyme boosts detection sensitivity through catalytic cleavage, enabling swift and specific identification of miRNA-21. This dual amplification strategy offers a highly sensitive, specific, and rapid alternative to traditional techniques, making it particularly well-suited for early-stage disease diagnosis. Key features • This protocol enables a sensitive detection of miRNA-21. • The technique employs an isothermal, enzyme-free bHCR process that can be carried out using standard laboratory equipment, eliminating the necessity for specialized instruments. • The entire protocol can be finalized in less than five hours, offering a swift and effective approach for high-throughput miRNA detection. • Minimal input RNA is needed for the protocol, and the sample preparation steps are straightforward.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5134"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669909/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142903671","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}

引用次数: 0

Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms. 免疫荧光原位检测光合生物TOR激酶活性。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5140

Ana P Lando, María A De Marco, Andrea C Cumino, Giselle M A Martínez-Noël

{"title":"Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms.","authors":"Ana P Lando, María A De Marco, Andrea C Cumino, Giselle M A Martínez-Noël","doi":"10.21769/BioProtoc.5140","DOIUrl":"10.21769/BioProtoc.5140","url":null,"abstract":"The target of rapamycin (TOR) is a central hub kinase that promotes growth and development in all eukaryote cells. TOR induces protein synthesis through the phosphorylation of the S6 kinase (S6K), which, in turn, phosphorylates ribosomal S6 protein (RPS6) increasing this anabolic process. Therefore, S6K and RPS6 phosphorylation are generally used as readouts of TOR activity. Protein phosphorylation levels are measured by a western blot (WB) technique using an antibody against one specific phosphosite in cell extracts. However, at the tissue/cell-specific level, there is a huge gap in plants due to the lack of alternative techniques for the evaluation of TOR activity as there are for other organisms such as mammals. Here, we describe an in vivo protocol to detect S6K phosphorylation in tissues/cells of model photosynthetic organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Our proposed method consists of the immunolocalization of a phosphorylated target of TOR kinase using a fluorescent secondary antibody by confocal microscopy. The protocol involves four main steps: tissue/cell fixation, permeabilization, and incubation with primary and secondary antibodies. It is an easy technique that allows handling different samples at the same time. In addition, different ultrastructural cell markers can also be used, such as for nucleus and cell wall detection, allowing a detailed analysis of cell morphology. To our knowledge, this is the first protocol to detect TOR activity in situ in photosynthetic organisms; we consider that it will pave the research on the TOR kinase, opening new possibilities to better understand its complex signaling. Key features • The protocol is an easy and non-destructive method to detect S6K phosphorylation at the cellular level for plants and algae. • First method for in situ immunolocalization of target proteins of TOR kinase in photosynthetic organisms.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5140"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669857/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904295","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}

引用次数: 0

Isolation of Viral Biofilms From HTLV-1 Chronically Infected T Cells and Integrity Analysis. HTLV-1慢性感染T细胞中病毒生物膜的分离及完整性分析

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5152

Coline Arone, Hélène Dutartre, Delphine Muriaux

{"title":"Isolation of Viral Biofilms From HTLV-1 Chronically Infected T Cells and Integrity Analysis.","authors":"Coline Arone, Hélène Dutartre, Delphine Muriaux","doi":"10.21769/BioProtoc.5152","DOIUrl":"10.21769/BioProtoc.5152","url":null,"abstract":"The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus that predominantly spreads through cell-to-cell contact due to the limited infectivity of cell-free viruses. Among various modes of intercellular transmission, HTLV-1 biofilms emerge as adhesive structures, polarized at the cell surface, which encapsulate virions within a protective matrix. This biofilm is supposed to facilitate simultaneous virion delivery during infection. Yet, the molecular and functional intricacies of viral biofilms remain largely unexplored, despite their pivotal role in understanding retroviral pathogenesis. In this study, we optimized a protocol to isolate HTLV-1 biofilms from chronically infected T cells, facilitating their structural and molecular characterization using proteomic and super-resolution microscopy analyses. This protocol involves cultivating HTLV-1 chronically infected T cells at high density to facilitate the natural detachment of viral biofilms into the supernatant. Then, employing successive centrifugations, the cells are separated from the detached biofilms, and these structures are pelleted at medium speed (10,000× g). This method circumvents the need for mechanical, chemical, or enzymatic biofilm detachment, bypasses the use of ultracentrifugation, and enables us to resuspend the biofilms in the appropriate buffer for subsequent analyses such as western blotting or super-resolution microscopy imaging as presented. Key features • Isolation of viral biofilms from HTLV-1 chronically infected T cells after 4 days of culture at high cellular density. • Structural analysis of viral biofilms using super-resolution microscopy techniques. • Experiments performed in vitro within a confined biosafety level 3 (BSL3) environment. • This protocol requires at least five days to complete.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5152"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669908/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904299","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}

引用次数: 0

Muscle Biopsy Sample Preparation and Proteomics Analysis Based on UHPLC-MS/MS. 基于UHPLC-MS/MS的肌肉活检样品制备及蛋白质组学分析。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5137

Jiawei Du, Jinghua Hou, Hezhang Yun, Yafeng Song

{"title":"Muscle Biopsy Sample Preparation and Proteomics Analysis Based on UHPLC-MS/MS.","authors":"Jiawei Du, Jinghua Hou, Hezhang Yun, Yafeng Song","doi":"10.21769/BioProtoc.5137","DOIUrl":"10.21769/BioProtoc.5137","url":null,"abstract":"Proteomics analysis is crucial for understanding the molecular mechanisms underlying muscle adaptations to different types of exercise, such as concentric and eccentric training. Traditional methods like two-dimensional gel electrophoresis and standard mass spectrometry have been used to analyze muscle protein content and modifications. This protocol details the preparation of muscle samples for proteomics analysis using ultra-high-performance liquid chromatography (UHPLC). It includes steps for muscle biopsy collection, protein extraction, digestion, and UHPLC-based analysis. The UHPLC method offers high-resolution separation of complex protein mixtures, providing more detailed and accurate proteomic profiles compared to conventional techniques. This protocol significantly enhances sensitivity, reproducibility, and efficiency, making it ideal for comprehensive muscle proteomics studies. Key features • Developed for analyzing muscle adaptations in response to concentric and eccentric training, applicable to various physiology exercise studies. • Utilizes UHPLC-MS/MS for high-resolution separation and detailed proteomic profiling. • Requires access to advanced UHPLC-MS/MS equipment and muscle biopsy collection tools. • The protocol can be completed within one week, including sample preparation and analysis.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5137"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669853/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904307","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}

引用次数: 0

Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay. 使用微粒体囊泡萌发试验评估 SREBP 激活情况

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5139

Mingfeng Xia, Tessa Edwards, Shunxing Rong

{"title":"Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay.","authors":"Mingfeng Xia, Tessa Edwards, Shunxing Rong","doi":"10.21769/BioProtoc.5139","DOIUrl":"10.21769/BioProtoc.5139","url":null,"abstract":"Sterol regulatory element binding proteins (SREBPs) are transcription factors that reside in the endoplasmic reticulum (ER) membrane as inactive precursors. To be active, SREBPs are translocated to the Golgi where the transcriptionally active N-terminus is cleaved and released to the nucleus to regulate gene expression. Nuclear SREBP levels can be determined by immunoblot analysis; however, this method can only determine the steady-state levels of nuclear SREBPs and does not capture the actual status of activation. The vesicle budding assay provides an alternative way to quantify the activation of SREBPs by monitoring the initiation of SREBP translocation from the ER to the Golgi through vesicles. Microsomal membranes isolated from the liver are incubated in a reaction buffer containing the necessary components to facilitate vesicle formation. Microsomal membranes and vesicles are isolated and SREBPs are quantified in each by immunoblot analysis. The amount of SREBPs found in the budded vesicles provides an assessment of the SREBP activation in the liver. Key features • This protocol describes a method to isolate budding vesicles from liver ER membranes. • The in vitro budding assay can be applied to investigate the movement of proteins from the ER to the Golgi. • This protocol was developed based on the procedures described previously with cultured cells [1-3].","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5139"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669852/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904236","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}

引用次数: 0

CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells. 基于CRISPR/ cas9的诱导多能干细胞精确基因组编辑方案

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5141

Avinash Singh, Swathy Babu, Marcus Phan, Shauna H Yuan

{"title":"CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells.","authors":"Avinash Singh, Swathy Babu, Marcus Phan, Shauna H Yuan","doi":"10.21769/BioProtoc.5141","DOIUrl":"10.21769/BioProtoc.5141","url":null,"abstract":"The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However, the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis, potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing, which will ultimately increase editing efficiency. In our study, we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines. Key features • The combination of p53 inhibition and pro-survival small molecules promotes cell survival and increases the efficiency of genome editing. • Genome editing can be completed in as little as 8 weeks for iPSCs, significantly reducing the total time required. • Achieves a homologous recombination rate of over 90% in human iPSCs.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5141"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669859/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904280","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}

引用次数: 0

Live Visualization of Calcified Bones in Zebrafish and Medaka Larvae and Juveniles Using Calcein and Alizarin Red S. 钙黄素和茜素红S对斑马鱼和鳉鱼幼鱼骨钙化的实时可视化研究。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5142

Rina Koita, Sae Oikawa, Taisei Tani, Masaru Matsuda, Akinori Kawamura

{"title":"Live Visualization of Calcified Bones in Zebrafish and Medaka Larvae and Juveniles Using Calcein and Alizarin Red S.","authors":"Rina Koita, Sae Oikawa, Taisei Tani, Masaru Matsuda, Akinori Kawamura","doi":"10.21769/BioProtoc.5142","DOIUrl":"10.21769/BioProtoc.5142","url":null,"abstract":"Zebrafish and medaka are valuable model vertebrates for genetic studies. The advent of CRISPR-Cas9 technology has greatly enhanced our capability to produce specific gene mutants in zebrafish and medaka. Analyzing the phenotypes of these mutants is essential for elucidating gene function, though such analyses often yield unexpected results. Consequently, providing researchers with accessible and cost-effective phenotype analysis methods is crucial. A prevalent technique for investigating calcified bone development in these species involves using transgenic fish that express fluorescent proteins labeling calcified bones; however, acquiring these fish and isolating appropriate crosses can be time-consuming. We present a comprehensive protocol for visualizing ossified bones in zebrafish and medaka larvae and juveniles using calcein and alizarin red S staining, which is both economical and efficient. This method, applicable to live specimens during the ossification of bones, avoids apparent alterations in skeletal morphology and allows for the use of different fluorescent dyes in conjunction with transgenic labeling, thus enhancing the analysis of developmental processes in calcifying bones, such as vertebrae and fin rays. Key features • The calcified bones of alive zebrafish and medaka larvae and juveniles can be visualized repeatedly using simple and inexpensive calcein and alizarin red S. • No need to use transgenic fish to visualize ossified bones, allowing for rapid analysis of bone phenotypes in mutants. • Double staining is possible in transgenic fish with reporter genes such as GFP and DsRed using alizarin red S or calcein, which exhibit different fluorescence. • Ossification processes of bones such as vertebrae, ribs, and fin rays can be analyzed in mutants.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5142"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669858/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904302","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}

引用次数: 0

Cryo-SEM Investigation of Chlorella Using Filter Paper as Substrate. 以滤纸为底物的小球藻低温扫描电镜研究。

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Bio-protocol Pub Date : 2024-12-20 DOI: 10.21769/BioProtoc.5143

Peng Wan, Meiyue Tao, Yumeng Zhou, Wenjun Han, Jianxia Wang, Jinghan Wang

{"title":"Cryo-SEM Investigation of Chlorella Using Filter Paper as Substrate.","authors":"Peng Wan, Meiyue Tao, Yumeng Zhou, Wenjun Han, Jianxia Wang, Jinghan Wang","doi":"10.21769/BioProtoc.5143","DOIUrl":"10.21769/BioProtoc.5143","url":null,"abstract":"Cryo-electron microscopy (cryo-EM) is a powerful technique capable of investigating samples in a hydrated state, compared to conventional high-vacuum electron microscopy that requires samples to be completely dry. During the drying process, numerous features and details may be lost due to damage caused by dehydration. Cryo-EM circumvents these problems by cryo-fixing the samples, thereby retaining the intact and original features of hydrated samples. This protocol describes a step-by-step cryo-scanning electron microscopy (cryo-SEM) experimental procedure with Chlorella sorokiniana as the subject. By employing filter paper as the sample substrate, we propose a simple and reliable method for cryo-fixation and freeze-fracture of Chlorella sorokiniana in water suspension. The advantage of using filter paper as a substrate lies in its ability to support a thin film of sample, enabling a cold knife to make a cut effortlessly and produce a clean freeze-fractured surface for SEM investigation. By following the approach described in this protocol, both the internal structure and surface morphology of Chlorella sorokiniana can be easily resolved with high quality. This protocol is highly versatile and can be applied to samples dispersed in water or solvents, including cyanobacterial cells, algal cells, and any kind of sample that can be adsorbed onto filter paper. Key features • Introducing a reliable way for ideal freeze-fracture of a water-suspended sample using filter paper as substrate. • Detailed step-by-step descriptions of the entire experiment, covering how to operate the instruments and including some practical experimental tips. Graphical overview.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 24","pages":"e5143"},"PeriodicalIF":1.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142904286","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}

引用次数: 0