Fixation and Immunolabeling of Brain Slices: SNAPSHOT Method

Dissing-Olesen, Lasse; MacVicar, Brian A.

doi:10.1002/0471142301.ns0123s71

Cited by 18 publications

(17 citation statements)

References 20 publications

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“…Some of the partially reconstructed slices were fixed using a new “hot” fixation method, SNAPSHOT (Dissing-Olesen and MacVicar, 2015). The ‘hot’ method involves immersing the brain slice in 4% paraformaldehyde pre-heated to 80 °C for 2 min.…”

Section: Resultsmentioning

confidence: 99%

“…One potential issue with the high-temperature fixation is that it can quench endogenous fluorescence (Dissing-Olesen and MacVicar, 2015), which in our case is needed to guide tissue trimming before and after embedding, as well as for ultrathin sectioning. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We routinely use microwave irradiation to speed up fixative penetration and chemical interaction, but it still requires 1 h of fixation to achieve well reproducible results. To improve time resolution, we used a new ‘hot’ fixation method (Dissing-Olesen and MacVicar, 2015). Control experiments confirmed that this method provides a rapid (less than 2.5 min) fixation of our samples without compromising tissue structure and antigenicity or quenching dye fluorescence.…”

Section: Discussionmentioning

confidence: 99%

“…Some of the partially reconstructed slices were fixed using a new “hot” fixation method, SNAPSHOT (Dissing-Olesen and MacVicar, 2015). This method results in rapid fixation of tissue, which is important when correlating anatomical features of the tissue with relatively fast physiological changes.…”

Section: Methodsmentioning

confidence: 99%

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Array tomography of physiologically-characterized CNS synapses

Valenzuela

Király

Liu

et al. 2016

Journal of Neuroscience Methods

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Background The ability to correlate plastic changes in synaptic physiology with changes in synaptic anatomy has been very limited in the central nervous system because of shortcomings in existing methods for recording the activity of specific CNS synapses and then identifying and studying the same individual synapses on an anatomical level. New method We introduce here a novel approach that combines two existing methods: paired neuron electrophysiological recording and array tomography, allowing for the detailed molecular and anatomical study of synapses with known physiological properties. Results The complete mapping of a neuronal pair allows determining the exact number of synapses in the pair and their location. We have found that the majority of close appositions between the presynaptic axon and the postsynaptic dendrite in the pair contain synaptic specializations. The average release probability of the synapses between the two neurons in the pair is low, below 0.2, consistent with previous studies of these connections. Other questions, such as receptor distribution within synapses, can be addressed more efficiently by identifying only a subset of synapses using targeted partial reconstructions. In addition, time sensitive events can be captured with fast chemical fixation. Comparison with existing methods Compared to existing methods, the present approach is the only one that can provide detailed molecular and anatomical information of electrophysiologically-characterized individual synapses. Conclusions This method will allow for addressing specific questions about the properties of identified CNS synapses, even when they are buried within a cloud of millions of other brain circuit elements.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Array tomography of physiologically-characterized CNS synapses

Valenzuela

Király

Liu

et al. 2016

Journal of Neuroscience Methods

View full text Add to dashboard Cite

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“…All the above processes may contribute to what has been defined as ‘ latent sensitization ’ in which the remission phase from chronic pain does not represent the healthy state (Marvizon et al, 2015; Walwyn et al, 2016). Thus patients may be more susceptible to the unveiling or recrudescence of pain symptoms and behavior.…”

Section: The Tipping Point: Neurobiological Processes Brain Dysfumentioning

confidence: 99%

Subliminal (latent) processing of pain and its evolution to conscious awareness

Borsook

Youssef

Barakat

et al. 2018

Neuroscience & Biobehavioral Reviews

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By unconscious or covert processing of pain we refer to nascent interactions that affect the eventual deliverance of pain awareness. Thus, internal processes (viz., repeated nociceptive events, inflammatory kindling, re-organization of brain networks, genetic) or external processes (viz., environment, socioeconomic levels, modulation of epigenetic status) contribute to enhancing or inhibiting the presentation of pain awareness. Here we put forward the notion that for many patients, ongoing sub-conscious changes in brain function are significant players in the eventual manifestation of chronic pain. In this review, we provide clinical examples of nascent or what we term pre-pain processes and the neurobiological mechanisms of how these changes may contribute to pain, but also potential opportunities to define the process for early therapeutic interventions.

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Live‐Cell Visualization of Calcium Flux in Vibratome‐Cut Thick Sections of Viable Tumor Tissue

2017

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This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein-coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment. The technical steps are performed in three sequential phases: (1) viable tissue sectioning; (2) fluorophore loading and tissue immobilization; and (3) live-cell confocal microscopy. This versatile method provides a straightforward platform for functional characterization of human tumors. © 2017 by John Wiley & Sons, Inc.

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Fixation and Immunolabeling of Brain Slices: SNAPSHOT Method

Cited by 18 publications

References 20 publications

Array tomography of physiologically-characterized CNS synapses

Array tomography of physiologically-characterized CNS synapses

Subliminal (latent) processing of pain and its evolution to conscious awareness

Live‐Cell Visualization of Calcium Flux in Vibratome‐Cut Thick Sections of Viable Tumor Tissue

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