Cold Spring Harbor protocols最新文献

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins. In this protocol, we describe use of the membrane fractionation kit to isolate membrane-bound proteins, followed by western blot analysis, to observe protein abundance. The protocol involves methods that require organ (or tissue) collection, followed by protein processing, and a 2-d western blot procedure.

This introduction reviews techniques used to examine the distribution and expression of gene transcripts and proteins in a variety of tissues/organs in the medically important global disease vector mosquito, Aedes aegypti Specifically, these methods allow the detection of cell-specific transcript expression by fluorescent in situ hybridization; facilitate immunohistochemical mapping of a protein of interest in whole-mount small tissue/organ samples; examine the subcellular localization of proteins, such as membrane transporters, through sectioning of paraffin-embedded tissue/organ samples; and finally, enable the efficient separation of cytosolic and membrane proteins for western blot analysis without the need for specialized equipment (e.g., ultracentrifuge) in the mosquito Ae. aegypti Such techniques are useful to help answer fundamental questions in mosquito scientific research including (but not limited to) the identification of specific cells in an organ responsible for expressing a receptor of particular interest and necessary for eliciting a response to exogenous signals, including hormones. Moreover, changes in the subcellular localization of specific targets of interest can be assessed both qualitatively and quantitatively, providing insight into transient or long-term physiologically relevant regulation necessary for activity under experimental treatments or varied internal (e.g., development) or external (e.g., environmental stress) factors that might be normally experienced by the organism.

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues. Here, we describe an IHC protocol for examining apical versus basolateral protein staining through sectioning tissue samples from fixed, embedded tissues (e.g., IHC-paraffin) and adding primary antibodies against a target protein. This IHC protocol provides a guide for tissue fixation, sectioning, and staining of tissue samples.

Neurons have a complex dendritic architecture that governs information flow through a circuit. Manual quantification of dendritic arbor morphometrics is time-consuming and can be inaccurate. Automated quantification systems such as DeTerm help to overcome these limitations. DeTerm is a software tool that automatically recognizes dendrite branch terminals with high precision. It uses an artificial neural network to label the terminals, count them, and provide each terminal's positional data. DeTerm can recognize the dendritic terminals of Drosophila dendritic arborization (da) neurons, and it can also examine other types of neurons, including mouse Purkinje cells. It is freely available and works on Mac, Windows, and Linux. Here, we describe the use of DeTerm.

Immunohistochemistry (IHC) is a powerful technique used for visualizing cellular components and determining the presence and/or location of proteins or other macromolecules in tissue samples. The classical IHC process involves the detection of epitopes using a highly specific primary antibody. This is followed by a secondary antibody that is coupled to a reporter molecule or fluorophore and capable of binding to the primary antibody and allowing for protein immunodetection. Although IHC does not routinely provide quantitative results compared to an enzyme-linked immunoassay or western blotting, it permits the localization of the proteins in intact tissues. This protocol describes an IHC assay for whole-body Aedes aegypti mosquito tissues that is used to detect small proteins, specifically neuropeptide hormones. This method is useful for protein detection in whole-mount preparations; however, cross-section IHC is recommended to determine if a protein is localized in the apical versus basolateral membrane of tissues/organs or to visualize immunological distribution in larger, more complex preparations.

Neurons receive, process, and integrate inputs. These operations are organized by dendrite arbor morphology, and the dendritic arborization (da) neurons of the Drosophila peripheral sensory nervous system are an excellent experimental model for examining the differentiation processes that build and shape the dendrite arbor. Studies in da neurons are enabled by a wealth of fly genetic tools that allow targeted neuron manipulation and labeling of the neuron's cytoskeletal or organellar components. Moreover, as da neuron dendrite arbors cover the body wall, they are highly accessible for live imaging analysis of arbor patterning. Here, we outline the structure and function of different da neuron types and give examples of how they are used to elucidate central mechanisms of dendritic arbor formation.

Live imaging approaches are essential for monitoring how neurons go through a coordinated series of differentiation steps in their native mechanical and chemical environment. These imaging approaches also allow the study of dynamic subcellular processes such as cytoskeleton remodeling and the movement of organelles. Drosophila dendritic arborization (da) neurons are a powerful experimental system for studying the dendrite arbor in live animals. da neurons are located on the internal surface of the body wall and, therefore, are easily accessible for imaging. Moreover, many genetic tools target da neurons to disrupt genes or proteins of interest and allow the investigator to visualize fluorescent markers and endogenously tagged proteins in the neurons. This protocol introduces methods for preparing and mounting intact Drosophila embryos, larvae, and pupae, allowing live imaging of dynamic cellular processes in da neurons.

Mosaic analysis with a repressible cell marker (MARCM) is used in Drosophila research to create labeled homozygous mutant clones of cells in an otherwise heterozygous fly. It allows the study of the effect of embryonically lethal genes and the determination of cell autonomy for a mutant phenotype. When used in dendritic arborization (da) neurons with a fluorescent protein targeted to the plasma membrane, MARCM allows the identification of homozygous mutant neurons and clear imaging of the dendrite arbor in both live and fixed preparations. Previous protocols that outlined experimental procedures to create MARCM clones in da neurons used a heat shock promoter to drive Flippase (FLP) expression; such an approach requires laborious embryo collection and heat shock steps, and it creates clones in other tissues besides the da neurons. The updated protocol described here outlines the use of FLP expression driven by a sensory organ precursor promoter (SOP-FLP); it requires no embryo collection or manipulation steps and creates clones exclusively in the peripheral sensory neuron lineage.

Drosophila dendritic arborization (da) neurons are a powerful model for studying neuronal differentiation and sensory functions. A general experimental strength of this model is the examination of the neurons in situ in the body wall. However, for some analyses, restricted access to the neurons in situ causes difficulty; da neuron cultures circumvent this. Here, we outline isolation and culture techniques for larval and pupal da neurons. Investigators can use these cultures to perform high-resolution imaging, quantitative immunohistochemistry, and electrophysiology.

Maize (Zea mays), also known as corn, is an important crop that plays a crucial role in global agriculture. The economic uses of maize are numerous, including for food, feed, fiber, and fuel. It has had a significant historical importance in research as well, with important discoveries made in maize regarding plant domestication, transposons, heterosis, genomics, and epigenetics. Unfortunately, environmental stresses cause substantial yield loss to maize crops each year. Yield losses are predicted to increase in future climate scenarios, posing a threat to food security and other sectors of the global economy. Developing efficient methods to study maize abiotic stress responses is a crucial step toward a more resilient and productive agricultural system. This review describes the importance of and methods for studying the effects of heat, drought, and nutrient deficiency on early developmental stages of maize grown in controlled environments. Studying the early effects of environmental stressors in controlled environments allows researchers to work with a variety of environmental conditions with low environmental variance, which can inform future field-based research. We highlight the current knowledge of physiological responses of maize to heat, drought, and nutrient stress; remaining knowledge gaps and challenges; and information on how standardized protocols can address these issues.