University of Georgia

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr exhibit morphological development comparable to embryos developing in utero.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

This protocol describes the complementary neuroimaging techniques of resting state structural connectivity, task-induced deactivation, and structural connectivity analyses to examine the default network in post-traumatic stress disorder. The use of synergistic methods could potentially lead to improved diagnostics and assessments of severity, outcome, and other relevant clinical factors.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

In this protocol, we outline the conceptual design elements and structural development of a glare acuity apparatus. Additionally, the design of a device for measuring positive dysphotopsia (halos, spokes) and two-point light thresholds is described.

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

A liquid-liquid extraction (LLE) system involving hollow-fiber membranes was developed to continuously and selectively extract medium-chain fatty acids (MCFAs) from the fermentation broth. The LLE system achieves high MCFA specificities from broths containing short-chain fatty acids and alcohols. Also, MCFAs are concentrated in a stripping solution to facilitate product recovery.