Most Cited —BIO-PROTOCOL

Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis: An Integrated Protocol

Authors: Alexandra R Crowe and Wei Yue, date: 12/20/2019, view: 13049, Q&A: 1

Semi-quantitative immunohistochemistry (IHC) is a powerful method for investigating protein expression and localization within tissues that involves using software, such as the freely available Fiji (ImageJ), to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed procedure on ...

Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe

Authors: Farzane Sivandzade, Aditya Bhalerao and Luca Cucullo, date: 01/05/2019, view: 29611, Q&A: 0

In recent years, fluorescent dyes have been frequently used for monitoring mitochondrial membrane potential to evaluate mitochondrial viability and function. However, the reproducibility of the results across laboratories strongly depends upon following well validated and reliable protocols along with the appropriate controls. Herein, we provide a ...

Detection and Analysis of Circular RNAs by RT-PCR

Authors: Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 25744, Q&A: 6

Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...

DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes

Authors: Rituraj Marwaha and Mahak Sharma, date: 10/05/2017, view: 23579, Q&A: 0

Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal ...

Polysome Fractionation to Analyze mRNA Distribution Profiles

Authors: Amaresh C. Panda, Jennifer L. Martindale and Myriam Gorospe, date: 02/05/2017, view: 28817, Q&A: 5

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that ...

Detection of Reactive Oxygen Species in Oryza sativa L. (Rice)

Authors: Navdeep Kaur, Isha Sharma, Kamal Kirat and Pratap Kumar Pati, date: 12/20/2016, view: 20692, Q&A: 2

Superoxide ions (O^2-) and hydrogen peroxide (H₂O₂) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O^2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H₂O₂ is qualitatively ...

Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation

Authors: Esteban A. Orellana and Andrea L. Kasinski, date: 11/05/2016, view: 45920, Q&A: 0

The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be ...

Protocol for Primary Microglial Culture Preparation

Authors: Hong Lian, Ethan Roy and Hui Zheng, date: 11/05/2016, view: 22769, Q&A: 0

Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse ...

BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry

Authors: Bo Qiu and M. Celeste Simon, date: 09/05/2016, view: 57873, Q&A: 0

Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis ...

Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei

Authors: Klaus Weisshart, Jörg Fuchs and Veit Schubert, date: 02/05/2016, view: 12283, Q&A: 0

RNA polymerase II (RNAPII) is the enzyme transcribing most of the eukaryotic protein-coding genes. Analysing the distribution and quantification of RNAPII can help understanding its function in interphase nuclei. Although several investigations in mammals indicate the organization of RNAPII in so-called ‘transcription factories’ (Jackson et al. ...

Collagen-induced Arthritis: A Model for Murine Autoimmune Arthritis

Authors: Kathryn M. Pietrosimone, Mengyao Jin, Brad Poston and Peng Liu, date: 10/20/2015, view: 14606, Q&A: 0

Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine ...

Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions

Authors: Ian Barr and Feng Guo, date: 09/20/2015, view: 30947, Q&A: 2

Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by ...

Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography

Authors: Miroslava Schaffer, Benjamin D. Engel, Tim Laugks, Julia Mahamid, Jürgen M. Plitzko and Wolfgang Baumeister, date: 09/05/2015, view: 21067, Q&A: 0

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of ...

Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria

Authors: Tomáš Zavřel, Maria A. Sinetova and Jan Červený, date: 05/05/2015, view: 33998, Q&A: 4

This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids concentration is often related to ...

Analysis of Protein Stability by the Cycloheximide Chase Assay

Authors: Shih-Han Kao, Wen-Lung Wang, Chi-Yuan Chen, Yih-Leong Chang, Yi-Ying Wu, Yi-Ting Wang, Shu-Ping Wang, Alexey I Nesvizhskii, Yu-Ju Chen, Tse-Ming Hong and Pan-Chyr Yang, date: 01/05/2015, view: 60893, Q&A: 1

Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a ...

Hypoxia Studies with Pimonidazole in vivo

Authors: Kristina Y. Aguilera and Rolf A. Brekken, date: 10/05/2014, view: 26035, Q&A: 0

Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including ...

Histochemical Detection of Superoxide and H₂O₂ Accumulation in Brassica juncea Seedlings

Authors: Deepak Kumar, Mohd Aslam Yusuf, Preeti Singh, Meryam Sardar and Neera Bhalla Sarin, date: 04/20/2014, view: 32489, Q&A: 3

Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism. Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors. Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins ...

Isolation of Mouse Embryo Fibroblasts

Authors: Marian E Durkin, Xiaolan Qian, Nicholas C Popescu and Douglas R Lowy, date: 09/20/2013, view: 18962, Q&A: 0

Preparation of primary cultures of embryo fibroblasts from genetically engineered mouse strains can provide a valuable resource for analyzing the consequences of genetic alterations at the cellular level. Mouse embryo fibroblasts (MEFs) have been particularly useful in cancer research, as they have facilitated the identification of the genetic ...

Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling

Authors: Zhijin Zhang and Rongfeng Huang, date: 07/20/2013, view: 32537, Q&A: 2

The protocol has four sub-protocols, which are about the measurement of malondialdehyde, chlorophyll proline, soluble sugar, and glutathione content, respectively, in Arabidopsis seedling by using spectrophotometer. These methods are simple, effective and reproducible, which will help the researchers who are not familiar with these ...

Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves

Authors: Arsalan Daudi and Jose A. O’Brien, date: 09/20/2012, view: 79235, Q&A: 23

In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi et al. ...

Fast Detection and Quantification of Interictal Spikes and Seizures in a Rodent Model of Epilepsy Using an Automated Algorithm