往期刊物2014
卷册: 4, 期号: 18
生物化学
Dimethylmethylene Blue Assay (DMMB)
二甲基亚甲基蓝试验(DMMB)
Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate and hyaluronic acid. The GAGs are heterogeneous molecules with great variability in molecular mass and both sulfation density and pattern. Spectrophotometric assays to measure the GAG content in biological fluids and tissue/cell extracts are valuable tools. The dye 1,9-dimethylmethylene is a thiazine chromotrope agent that presents a change in the absorption spectrum due to the induction of metachromasia when bound to sulfated GAGs enabling rapid detection of GAGs in solution (Whitley et al., 1989; Chandrasekhar et al., 1987; Farndale et al., 1982). Moreover, there is a window in which a linear curve may be drawn (approximately between 0.5-5 μg of GAGs) enabling the quantification of GAGs in solution.
Protein-lipid Interaction Analysis by Surface Plasmon Resonance (SPR)
表面等离子共振 (SPR)法分析蛋白脂质的相互作用
Interactions of lipids with proteins are essential events in the framework of biological membranes. Assessment of the affinity and specificity of protein-lipid binding can give useful information to elucidate cell membrane functions. Surface Plasmon Resonance (SPR) is a powerful technology to study macromolecular interactions, allowing direct and rapid determination of association and dissociation rates using small amounts of samples. An extensive range of binding analyses can be performed by SPR such as protein–protein, protein–membrane (lipids), protein–carbohydrate, protein–nucleic acid and even protein-small molecules. This protocol describes the binding of an antimicrobial protein (used as ligand) to a lipopolysaccharide (LPS) (used as analyte) after immobilization onto a CM sensor chip by amine coupling.
免疫学
Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue
从CNS组织分离单核细胞及FACS分析
Immune cells, such as microglia are resident in the brain and spinal cord of normal mice and humans. Furthermore, macrophages, dendritic cells, T cells, B cells and NK cells infiltrate the CNS during certain infections or in neurodegenerative/neuroinflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) (a model for multiple sclerosis) or Alzheimer’s disease (Sutton et al., 2009; Browne et al., 2013). Infiltrating cells can be identified using immunohistological staining of sections from brain or spinal cords. However, more detailed phenotypic and functional analysis is possible following isolation of the immune cells from the CNS of normal or diseased mice. Purification of mononuclear cells from brain or spinal cord is dependent on perfusing the mouse to ensure removal of the blood from the CNS tissue, prior to dissociating the tissue and purification of the mononuclear cells on a percoll gradient. The technique provides single cell suspensions with cells of high viability that are suitable for FACS analysis or limited functional studies. The yields are usually low from the normal mouse brain or spinal cord, but higher from mice with EAE or CNS infection. When combined with intracellular cytokine staining and FACS, this technique is particularly useful for analysis of the pathogenic T cells (Th17 and Th1 cells) and their regulation/modulation in EAE.
Generation of Aβ-specific T cell lines and in vivo Transfer
β-淀粉样蛋白（Aβ）特异性T淋巴细胞系的建立和过继转移
Amyloid-β (Aβ)-containing plaques accumulate in the brains of patients with Alzheimer’s disease (AD). Studies in transgenic mice which over-express amyloid precursor protein and presenilin 1 (APP/PS1 mice) have suggested that T cells that infiltrate the brain may influence the development of Aβ plaques and associated cognitive dysfuncation. Active immunization with Aβ peptides and adjuvants has been evaluated as a therapy for AD, based on the premise that it induces Aβ-specific antibodies that may help to clear the Aβ plaques. However, immunization with Aβ peptides and adjuvants also promotes the development of Aβ-specific T cells (McQuillan et al., 2010) and there is evidence that Aβ-specific T cell may influence the development of Aβ plaques and disease progression in AD patients. In the mouse model, Aβ-specific T cells that secrete IFN-γ (Th1 cells) have been shown to enhance the plaque burden (Browne et al., 2013). Adoptive transfer of Aβ-specific T cells that have been polarized in vitro to Th1, Th2, Th17 or Treg cells can be used to examine the function of these cells in vivo.
微生物学
Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages
庆大霉素保护试验测定细菌在巨噬细胞中的存活能力
Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.
In vitro Transcription (IVT) and tRNA Binding Assay
体外转录（IVT）和tRNA结合试验
This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The first part of the protocol also describes the in vitro transcription and labeling of the tRNA.
Activity Assays for Bacteriophage Endolysin PlyPy
噬菌体细胞内溶素PlyPy的活性分析
Bacterial viruses (bacteriophages) escape and kill their host by degrading the bacterial peptidoglycan layer through the mechanism of enzymes called endolysins: peptidoglycan degrading enzymes. The method included here is useful for the initial characterization of any endolysin, regardless of the specific catalytic domain (as long as the activity results in a reduction in the optical density), in order to determine its optimal enzymatic (lytic) activity. This protocol is specific for the Streptococcus pyogenes phage endolysin PlyPy, but can be adapted for any peptidoglycan degrading enzyme.
Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein
白色念珠菌中响应抗真菌蛋白Hog1磷酸化水平的检测
It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans (C. albicans) MAPK Hog1 by monitoring its phosphorylation in response to an antifungal protein.
Protocol for Biotin Bioassay-based Cross Feeding
基于生物测定的生物素交叉喂养法
Biotin bioassay-based cross-feeding experiments were performed to elucidate the effect on biotin production by bioRbme expression in Agrobacterium tumefaciens (A. tumefaciens) (Feng et al., 2013). The indicator strain used here is the biotin auxotrophic strain of Escherichia coli (E. coli), ER90 (ΔbioF bioC bioD), which was cross-fed by A. tumefaciens species (Feng et al., 2013a). The biotin-free M9 minimal medium plates were formulated as described by other and our research groups (Feng et al., 2013b; Lin et al., 2010; del et al., 1979). Of note, 0.01% (w/v) the redox indicator 2, 3, 5-triphenyl tetrazolium chloride (TTC) was supplemented into the above media. Consequently, biotin generation/production was observed via the reduction of TTC to the insoluble red formazan which is due to the ER90 growth fed by A. tumefaciens strains (Feng et al., 2014). Detailed procedures are described as follows.
神经科学
Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections
自由浮动振动切片机中组织切片的组合原位杂交/免疫组织化学法(ISH/IH)
In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain. Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3’-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples of procedure results, please see images published in Lopez et al.. (2012).
植物科学
Perls Staining for Histochemical Detection of Iron in Plant Samples
普鲁士蓝染色用于植物样品中铁的组织化学检测
Visualization of iron (Fe) localization in plants has greatly enhanced our understanding of plant Fe homeostasis. One of the relatively simple and yet powerful techniques is the classical Perls blue stain (Perls, 1867). The technique is based on the conversion of ferrocyanide to insoluble crystals of Prussian blue in the presence of Fe3+ under acidic conditions. It has been extensively used in animal and human histology (Meguro et al., 2007) and has recently gained popularity in plant research. For specific purposes, Fe signals may be additionally enhanced in the 3,3’-diaminobenzidine tetrahydrochloride (DAB) intensification procedure (Meguro et al., 2007). It has been demonstrated that this intensification results in the detection of both Fe2+ and Fe3+ ions (Roschzttardtz et al., 2009). The method has been successfully applied at the whole plant, organ and subcellular levels, both with (Roschzttardtz et al., 2011; Schuler et al., 2012; Roschzttardtz et al., 2013; Ivanov et al., 2014) and without intensification (Stacey et al., 2008; Long et al., 2010).Here, we present a full Perls staining and DAB intensification protocol, the way it is performed in our lab (Ivanov et al., 2014).
Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves
从瞬时转化的本氏烟草中提取叶绿体蛋白质
This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis. Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005).The chloroplast isolation of the transformed leaves is based with modifications on Romeis et al. (2001), and includes mechanical breakage of cell wall and membranes, the removal of unbroken tissue by filtration and the separation of intact chloroplasts by centrifugation through a Percoll layer.
Thin Sections of Technovit 7100 Resin of Rice Endosperm and Staining
采用Technovit 7100树脂制备水稻胚乳薄切片并染色
Starch is a biologically and commercially important carbohydrate that is accumulated in plant storage organs, such as seed endosperm. Starch is water-insoluble and forms transparent grains, referred to as starch grains (SGs). SGs are easily stained by iodine solution and can be observed under a normal microscope. Technovit 7100 resin is suitable for preparation of thin sections from endosperm. The thin sections and iodine staining can visualize SGs clearly inside the endosperm cell. This protocol provides the procedures to prepare thin sections of Technovit 7100 resin from rice endosperm. It can also be applicable to the seeds of other plant species.Figure 1. Iodine-stained Technovit thin sections of matured endosperm cells in rice and barley. Starch grains (SGs) can be visualized as violet particles.