Bio-protocol

Cryo-scanning electron microscopy (cryo-SEM) was first introduced for scientific use in the 1980s. Since then, cryo-SEM has become a routine technique for studying the surfaces and internal structures of biological samples with a high water content. In contrast to traditional SEM, cryo-SEM requires no sample pretreatment processes; thus, we can obtain the most authentic images of the sample shape and structure. Cryo-SEM has two main steps: cryoprocessing of samples and scanning electron microscopy (SEM) observation. The cryoprocessing step includes preparation of the cooled slushing station, cooling of the preparation chamber, sample preparation, and sputtering. The sample is then transferred to an SEM cold stage for observation. We used cryo-SEM to study rice root hair tissues, but the methods and protocols can be applied to other root systems. This protocol optimizes the two key operation steps of reducing the humidity in the growth chamber and previewing the samples before sputtering and can more quickly obtain high-quality images.

Haiting Yan

Yue Wang

Jingrong Zhang

Xinru Cui

Jiasong Wu

Jie Zhou

Yuan Chen

Jia Lu

Ruiyang Guo

Maggie Ou

Hongxu Lai

Zhiming Yu

BIO-PROTOCOL

Rice Root Hair Phenotypes Imaged by Cryo-SEM

水稻根毛表型通过冷冻扫描电子显微镜成像

[Abstract] Here, we describe a simple and efficient tissue clearing method, CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis), which was first introduced in 2014 (Susaki et al ., 2014). In the present study, the hemispheres of wild-type adult mice were cleared by standard procedures and were browning free with good transparency and no significant changes in tissue shape or volume over a short time frame. Further, in combination with immunofluorescence staining, 3D high-resolution images of tyrosine hydroxylase-positive dopaminergic neurons in the substantina striatum region were obtained by rotating-disc confocal microscopy, in addition to images of mouse cortical and hippocampal neurons. Considering the limitations of existing brain tissue removal methods, such as CLARITY, 3DISCO, and SeeDB, CUBIC boasts simple operation, high fluorescent protein preservation, and good antibody penetration. Moreover, CUBIC variants and optimizations are available for the 3D imaging of rodent, large primate, and human tissues to meet researchers' specific requirements, which could make it a popular option among research labs. Our study demonstrates the use of the CUBIC protocol for understanding brain structure and function in model animals with neurological diseases. Flowchart of the CUBIC clearing and imaging protocol. A. Tissue preparation. The adult mouse was first perfused with cold PBS/PFA solution. The harvested brain tissue was fixed with 4% PFA on day 0. B. Tissue clearing. The tissue was incubated with 1/2-water-diluted CUBIC-1, resulting in the removal of hemochrome and lipid membranes. It usually takes 1-6 days to become more transparent. C. Immunostaining. Tissues (2-mm brain slice) were blocked and then stained with directly conjugated anti-TH diluent (1:100) and FITC diluent (1:500) on days 7-9. D. RI matching. Tissues were immersed in 1/2-PBS-diluted CUBIC-2 on day 10 to match the refractive index (RI). Following immersion in imaging oil for 2 h, the cellular position within tissues was photographed by confocal microscopy.

连响 罗

Xiaoyu Chen

Ruijian Wu

Han Wang

Hui Luo

Clearing of the Mouse Brain for Optical Imaging Using CUBIC

使用CUBIC清除小鼠大脑进行光学成像

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production of transcription- and replication-component virus-like-particles (trVLPs) by genetic transcomplementation. The system consists of two parts: SARS-CoV-2 GFP/ΔN genomic RNA, in which the nucleocapsid (N) gene, a critical gene for virion packaging, is replaced by a GFP reporter gene; and a packaging cell line for ectopic expression of N (Caco-2-N). The complete viral life cycle can be recapitulated and confined to Caco-2-N cells, with GFP positivity serving as a surrogate readout for viral infection. In addition, we utilized an intein-mediated protein splicing technique to split the N gene into two independent vectors and generated the Caco-2-Nintein cells as a packaging cell line to further enhance the security of this cell culture model. Altogether, this system provides for a safe and convenient method to produce trVLPs in BSL-2 laboratories. These trVLPs can be modified to incorporate desired mutations, permitting high-throughput screening of antiviral compounds and evaluation of neutralizing antibodies. This protocol describes the details of the trVLP cell culture model to make SARS-CoV-2 research more readily accessible.

Yanying Yu

Xiaohui Ju

Qiang Ding

A Nucleocapsid-based Transcomplementation Cell Culture System of SARS-CoV-2 to Recapitulate the Complete Viral Life Cycle

基于核衣壳的SARS-CoV-2转补体细胞培养系统以重现完整的病毒生命周期

We describe a protocol to measure the electrolyte leakage from plant tissues, resulting from loss of cell membrane integrity, which is a common definition of cell death. This simple protocol is designed to measure the electrolyte leakage from a tissue sample over a time course, so that the extent of cell death in the tissue can be monitored dynamically. In addition, it is easy to handle many tissue samples in parallel, which allows a high level of biological replication. Although the protocol is exemplified by cell death in

Noriyuki Hatsugai

Fumiaki Katagiri

Quantification of Plant Cell Death by Electrolyte Leakage Assay

植物细胞死亡的电解质渗漏法测定

Exercise induces beneficial systemic adaptations that reduce the incidence of age-related diseases.However, the molecular pathways that elicit these adaptations are not well understood.Understanding the molecular mechanisms that underlie the exercise response can lead to widely beneficial therapies.Large populations, relatively short lifespan, and easily modifiable genetics make Drosophila a well-suited model system for complex, longitudinal studies.We have developed an enforced climbing apparatus for Drosophila, known as the Power Tower, for the study of systemic exercise adaptations.The Power Tower takes advantage of the fly's natural instinct for negative geotaxis, an innate behavior to run upwards after being tapped to the bottom of their vial.Flies will continuously run either to the point of exhaustion or until the machine is turned off, whichever comes first.After 3 weeks of exercise, male Drosophila adapt to training with a number of conserved, easily quantifiable physiological improvements similar to those seen in mammalian models and humans.Here, we describe a useful endurance training protocol and a suite of post-training assessments that effectively quantify training effects.

Deena Damschroder

Tyler Cobb

Alyson Sujkowski

Robert Wessells

Drosophila Endurance Training and Assessment of Its Effects on Systemic Adaptations

果蝇耐力训练及其对系统性适应的影响评估

Key features• Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment.• Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.

Lauren E- Eyssen

Siva Ramadurai

Sahar Abdelkarim

Imogen Buckle

Katy Cornish

Hong Lin

A Jones

Gary Stephens

Raymond Owens

From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs

从羊驼到纳米抗体：功能化VHHs的简化工作流程

is an opportunistic pathogen that may cause serious infections in an immunocompromised host.

Pirjo Pärnänen

Timo Sorsa

Taina Tervahartiala

Pirjo Nikula-Ijäs

Preparation and Purification of β-1,3-glucan-Linked &lt;em&gt;Candida glabrata&lt;/em&gt; Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay

β-1,3-葡聚糖连接的<em>酿酒酵母</em>细胞壁蛋白酶的制备与纯化：离子交换色谱、凝胶过滤和MDPF-明胶-酶谱分析

Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells. Key features • Protocol for microfabrication of confinement devices. • Single-cell dynamic confinement coupled with high-resolution microscopy. • Static cell confinement protocol that can be combined with super-resolution STED microscopy. • Analysis of the mechanical control of mitotic entry in a time-resolved manner.

Margarida Dantas

Débora Vareiro

Jorge G Ferreira

Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup

剖析细胞限制装置对有丝分裂进入的力学调控

Mengya Jia

Dongqin Ding

Xiaohua Liu

Haixing Li

Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking

用于基因组步移的部分重叠引物PCR鉴定未知侧翼DNA的方案

Rui Zhou

Jiabin Pan

Xiang-Dong Li

Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST)

使用微量热泳动技术测定肌球蛋白-5a与其货物蛋白相互作用的解离常数

短名	Bio. Protoc.
Journal Impact	0.95
国际分区	BIOLOGY(Q3)

ISSN	2331-8325
h-index	18

涉及主题	life sciencesprotocolsmolecular biologybiochemistrybiologycell biology
出版信息	出版商: Bio-protocol LLC，出版周期: ，期刊类型: journal，开源期刊: 是
基本数据	创刊年份: 2018，原创研究文献占比: 100.00%，自引率:， Gold OA占比: 14.87%
期刊费用	$1200详情
平均审稿周期	12 weeks

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