University of Miami Miller School of Medicine

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

This article describes three methods for the introduction of materials into murine neonates. We demonstrate (a) intravenous injection in ≤1 day old neonates, (b) subcutaneous and intraperitoneal injection into 7 day old neonates, and (c) orogastric infusion into 7 day old neonates.

A challenge for proving treatment efficacy for cognitive impairments in schizophrenia is finding the optimizing measurement of skills related to everyday functioning.  The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is an interactive gaming based computerized measure aimed at skills associated with everyday functioning, including baseline impairments and treatment related changes.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets’ microenvironment in vitro and, potentially, in vivo settings.

Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field electroretinogram for infants and children under sedation or general anesthesia in the operating room setting.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each other, mostly using simultaneous-recording signals of brains (namely hyperscanning) with fNIRS. In fNIRS hyperscanning studies, IBS has been commonly assessed through the wavelet transform coherence (WTC) method because of its advantage on expanding time series into time-frequency space where oscillations can be seen in a highly intuitive way. The observed IBS can be further validated via the permutation-based random pairing of the trial, partner, and condition. Here, a protocol is presented to describe how to obtain brain signals via fNIRS technology, calculate IBS through the WTC method, and validate IBS by permutation in a hyperscanning study. Further, we discuss the critical issues when using the above methods, including the choice of fNIRS signals, methods of data preprocessing, and optional parameters of computations. In summary, using the WTC method and permutation is a potentially standard pipeline for analyzing IBS in fNIRS hyperscanning studies, contributing to both the reproducibility and reliability of IBS.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.