Immunostaining of Voltage-Gated Ion Channels in Cell Lines and Neurons – Key Concepts and Potential Pitfalls

Bocksteins, Elke; Shepherd, Andrew J.; Mohapatra, Durga P.; Snyders, Dirk J.

doi:10.5772/34817

Cited by 3 publications

(3 citation statements)

References 31 publications

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“…When applied to fixed tissue specimens, optimal results in IHC depend greatly on two basic elements: optimal fixation and tissue preservation (Schneider Gasser et al, 2006 ). Of the main fixatives generally employed in IHC (glutaraldehyde, paraformaldehyde, methanol/acetone), paraformaldehyde at various concentrations is widely used as it provides the most straightforward and rapid fixative method to expose antibody epitope(s) without compromising cell/tissue morphology (Bocksteins et al, 2012 ). However, detecting proteins in fine sub-cellular structures could be challenging using conventional fixatives (Schneider Gasser et al, 2006 ; Lorincz and Nusser, 2008b , 2010 ; Christensen et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

Alshammari

Laezza

2016

Front. Cell. Neurosci.

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The axonal initial segment (AIS) is the subcellular compartment required for initiation of the action potential in neurons. Scaffolding and regulatory proteins at the AIS cluster with ion channels ensuring the integrity of electrical signaling. Interference with the configuration of this protein network can lead to profound effects on neuronal polarity, excitability, cell-to-cell connectivity and brain circuit plasticity. As such, the ability to visualize AIS components with precision provides an invaluable opportunity for parsing out key molecular determinants of neuronal function. Fluorescence-based immunolabeling is a sensitive method for morphological and molecular characterization of fine structures in neurons. Yet, even when combined with confocal microscopy, detection of AIS elements with immunofluorescence has been limited by the loss of antigenicity caused by fixative materials. This technical barrier has posed significant limitations in detecting AIS components alone or in combination with other markers. Here, we designed improved protocols targeted to confocal immunofluorescence detection of the AIS marker fibroblast growth factor 14 (FGF14) in combination with the cytoskeletal-associated protein Ankyrin-G, the scaffolding protein βIV-spectrin, voltage-gated Na+ (Nav) channels (especially the Nav1.6 isoform) and critical cell type-specific neuronal markers such as parvalbumin, calbindin, and NeuN in the mouse brain. Notably, we demonstrate that intracardiac perfusion of animals with a commercially available solution containing 1% formaldehyde and 0.5% methanol, followed by brief fixation with cold acetone is an optimal and sensitive protocol for FGF14 and other AIS marker detection that guarantees excellent tissue integrity. With variations in the procedure, we also significantly improved the detection of Nav1.6, a Nav isoform known for its fixative-sensitivity. Overall, this study provides an ensemble of immunohistochemical recipes that permit excellent staining of otherwise invisible molecules within well-preserved tissue architecture. While improving the specific investigation of AIS physiology and cell biology, our thorough study can also serve as a roadmap for optimizing immunodetection of other fixative-sensitive proteins expanding the repertoire of enabling methods for brain studies.

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Section: Introductionmentioning

confidence: 99%

Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

Alshammari

Laezza

2016

Front. Cell. Neurosci.

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“…TRPC5 staining mostly appeared diffuse over the whole cell soma, and punctate with dark clusters at the edge of the cell (Bocksteins et al, 2012;Schaefer et al, 2000) (Figure 1f,g).…”

Section: Staining Characteristicsmentioning

confidence: 99%

Novel localizations of TRPC5 channels suggest novel and unexplored roles: A study in the chick embryo brain

Ahmed

Liu

Yip

et al. 2021

Developmental Neurobiology

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Mammalian TRPC5 channels are predominantly expressed in the brain, where they increase intracellular Ca 2+ and induce depolarization. Because they augment presynaptic vesicle release, cause persistent neural activity, and show constitutive activity, TRPC5s could play a functional role in late developmental brain events. We used immunohistochemistry to examine TRPC5 in the chick embryo brain between 8 and 20 days of incubation, and provide the first detailed description of their distribution in birds and in the whole brain of any animal species. Stained areas substantially increased between E8 and E16, and staining intensity in many areas peaked at E16, a time when chick brains first show organized patterns of whole-brain metabolic activation like what is seen consistently after hatching. Areas showing cell soma staining match areas showing Trpc5 mRNA or protein in adult rodents (cerebral cortex, hippocampus, amygdala, cerebellar Purkinje cells). Chick embryos show protein staining in the optic tectum, cerebellar nuclei, and several brainstem nuclei; equivalent areas in the Allen Institute mouse maps express Trpc5 mRNA. The strongest cell soma staining was found in a dorsal hypothalamic area (matching a group of parvicellular arginine vasotocin neurons and a pallial amygdalohypothalamic cell corridor) and the vagal motor complex. Purkinje cells showed strong dendritic staining at E20. Unexpectedly, we also describe neurite staining in the septum, several hypothalamic nuclei, and a paramedian raphe area; the strongest neurite staining was in the median eminence. These novel localizations suggest new unexplored TRPC5 functions, and possible roles in late embryonic brain development.

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“…The development of fluorescent labeling techniques allowed visualization of ion channel dynamics at the plasma membrane that provides complementary measurements to biochemical assays, with improved mechanistic insight . However, designing genetically encoded tagged ion channels is difficult due to potential interference with native channel function, which has greatly hindered the development of ion channel assays with genetic chemical labels . Given the limited creation of ion channel FP‐fusions or chemical labeling choices in general, ion channel FAP‐fusion constructs that exhibit native function represent a valuable tool option for labeling cell surface ion channels to assess trafficking and changes in trafficking associated with altered ionic transport.…”

Section: Cell Surface Protein Trafficking Measurements: Ion Channelsmentioning

confidence: 99%

Fluorogen activating protein toolset for protein trafficking measurements

Perkins

Bruchez

2020

Traffic

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Assessing protein trafficking in live cells is a core strength of fluorogen activating protein technology. The rapid and specific labeling of a tagged protein of interest, intracellular or surface, with a variety of fluorogenic dye derivatives creates a toolset suitable for scalable, dynamic multi‐color fluorescence experiments and direct quantitative trafficking‐related measurements in single cells and cell populations, using fluorometry, flow cytometry and microscopy.

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Immunostaining of Voltage-Gated Ion Channels in Cell Lines and Neurons – Key Concepts and Potential Pitfalls

Cited by 3 publications

References 31 publications

Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

Novel localizations of TRPC5 channels suggest novel and unexplored roles: A study in the chick embryo brain

Fluorogen activating protein toolset for protein trafficking measurements

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