New York University School of Medicine

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Non-invasive imaging of the brain vasculature’s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Here, based on a clinician’s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

Here we present PyDesigner, a Python-based diffusion magnetic resonance imaging (dMRI) processing pipeline capable of correcting for typical dMRI artifacts and producing diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), fiber ball imaging (FBI), microstructure modeling (white matter integrity [WMTI] and fiber ball white matter [FBWM]), and tractography outputs.