Ex Vivo Neuroimaging Techniques in Rodents
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Questions and Answers

What is the purpose of iDISCO in the study of the mouse brain?

To analyze the entire mouse brain vasculature and investigate effects of ischemic stroke.

Describe the brain modifications observed three weeks after an ischemic stroke according to the reconstruction.

There was a missing ventral branch in the middle cerebral artery and remodeled anastomosis with dorsal arteries, showing reorientation of vessels towards the stroked site.

What role does the EyeDISCO protocol serve in ocular research?

It is used for whole eye clearing to investigate the role of Dcc in optic nerve guidance during development.

What techniques are employed in the 3D rendering of brains noted in the EyeDISCO section?

<p>Anterograde axon tracing using AlexaFluor-555 and/or AlexaFluor-647-conjugated cholera toxin β subunit is used for visualization.</p> Signup and view all the answers

What are the key brain regions labeled in the 3D-rendered images of the EyeDISCO study?

<p>The key regions include the optic chiasm, lateral geniculate nucleus, and superior colliculus.</p> Signup and view all the answers

What is the purpose of incubating with a primary antibody in immunohistochemistry?

<p>The primary antibody binds to the specific antigen in the sample.</p> Signup and view all the answers

Identify one common detection method used in immunohistochemistry.

<p>Colorimetric detection using HRP enzyme and DAB/H2O2 substrate.</p> Signup and view all the answers

What is the role of the secondary antibody in immunohistochemistry?

<p>The secondary antibody binds to the primary antibody, amplifying the signal for detection.</p> Signup and view all the answers

What does the blocking step in the immunohistochemistry protocol achieve?

<p>Blocking prevents non-specific binding of antibodies to the tissue.</p> Signup and view all the answers

Name a fluorophore commonly used in fluorescent detection in immunohistochemistry.

<p>Alexa Fluor is a widely used fluorophore.</p> Signup and view all the answers

Why is it important to have a labeled secondary antibody?

<p>A labeled secondary antibody provides a detectable signal to visualize the primary antibody-antigen complex.</p> Signup and view all the answers

What is the general duration of a typical immunohistochemistry protocol?

<p>The protocol typically spans over 2 days.</p> Signup and view all the answers

Describe the role of the enzyme in colorimetric detection methods.

<p>The enzyme catalyzes a reaction that produces a color change in the presence of its substrate.</p> Signup and view all the answers

What technique does iDISCO+ utilize to visualize amyloid-β plaques?

<p>iDISCO+ utilizes optical clearing and 3D imaging techniques to visualize amyloid-β plaques.</p> Signup and view all the answers

How does the 3D complexity of amyloid-β plaques differ between humans and mice?

<p>Amyloid-β plaques exhibit greater 3D complexity in humans compared to mice.</p> Signup and view all the answers

What are the roles of detergents and chemicals in the iDISCO+ process?

<p>Detergents and chemicals are used for permeabilization to facilitate antibody entrance into the tissue.</p> Signup and view all the answers

What is the purpose of bleaching in the iDISCO+ technique?

<p>Bleaching is employed to eliminate autofluorescence from the samples.</p> Signup and view all the answers

What antibodies are used to label beta amyloid plaques in the cortex according to the presented videos?

<p>Anti-beta amyloid antibodies (PAb #10) are used to label beta amyloid plaques.</p> Signup and view all the answers

How does the iDISCO+ technology enhance the observation of microglia in relation to amyloid plaques?

<p>iDISCO+ facilitates the visualization of microglia enveloping the plaques using anti-Iba1 antibodies.</p> Signup and view all the answers

In the iDISCO+ technique, what additional structures are labeled alongside amyloid plaques in the first video?

<p>Cell nuclei are labeled with TO-PRO-3 alongside beta amyloid plaques in the cortex.</p> Signup and view all the answers

What does the surface rendering technique in iDISCO+ help to isolate?

<p>Surface rendering helps to isolate beta amyloid deposits along blood vessels.</p> Signup and view all the answers

What is the main difference between ex vivo and in vivo neuroimaging techniques?

<p>Ex vivo techniques involve analyzing tissues after the organism's death, while in vivo techniques observe brain function in living animals.</p> Signup and view all the answers

Name one histochemical staining technique and its purpose.

<p>Hematoxylin/eosin is a histochemical staining technique used to visualize brain morphology.</p> Signup and view all the answers

What is the purpose of using anesthetics and analgesia during animal preparation for ex vivo neuroimaging?

<p>Anesthetics and analgesia minimize pain and distress in the animals prior to euthanasia for brain study.</p> Signup and view all the answers

Describe a method used for making brain sections.

<p>A cryostat is used to cut freezing thin sections of the brain for analysis.</p> Signup and view all the answers

What is the purpose of 3DISCO and similar techniques?

<p>3DISCO is used for whole brain clearing, allowing for visualization of brain structures in three dimensions.</p> Signup and view all the answers

Why might a researcher choose to use immunohistochemistry in their study?

<p>Immunohistochemistry allows researchers to visualize specific proteins or markers within tissue samples.</p> Signup and view all the answers

What is the significance of utilizing Nissl staining in brain morphology studies?

<p>Nissl staining helps in quantifying neurons and assessing brain morphology by highlighting cellular structures.</p> Signup and view all the answers

What role does fixation play in the preparation of brain samples for imaging?

<p>Fixation preserves the brain's cellular architecture and prevents degradation during subsequent analysis.</p> Signup and view all the answers

What is the primary purpose of the uDISCO technique?

<p>The primary purpose of uDISCO is to enable ultimate three-dimensional imaging of solvent cleared organs.</p> Signup and view all the answers

How does the optimization of the 3DISCO protocol utilize tissue shrinkage?

<p>The optimization takes advantage of tissue shrinkage to facilitate imaging of larger structures than before.</p> Signup and view all the answers

In the context of uDISCO, what does 'whole body clearing' refer to?

<p>Whole body clearing refers to the process of making an entire organism transparent for comprehensive imaging.</p> Signup and view all the answers

What is the significance of tracing individual axons in the spinal cord of the sample in uDISCO?

<p>Tracing individual axons allows for the determination of their entire trajectories within the central nervous system.</p> Signup and view all the answers

What additional steps are included in the iDISCO protocol compared to standard immunohistochemistry?

<p>The additional steps in iDISCO include permeabilization, bleaching, and blocking, followed by antibody incubations.</p> Signup and view all the answers

What role does methanol play in the iDISCO protocol?

<p>Methanol is used to dehydrate the brain tissue during the clearing process in iDISCO.</p> Signup and view all the answers

Which fluorescently labeled proteins does uDISCO utilize for imaging?

<p>uDISCO uses only endogenous fluorescently labeled proteins for imaging purposes.</p> Signup and view all the answers

What are the two primary components involved in the clearing process of the iDISCO protocol?

<p>The two primary components involved in the clearing process are methanol and dichloromethane.</p> Signup and view all the answers

What is doublecortin (DCX) and its significance in studying neurons?

<p>Doublecortin (DCX) is a marker for newly-formed neurons, indicating neurogenesis.</p> Signup and view all the answers

What was the main finding of the study regarding stroke and neuron production?

<p>The study found that stroke resulted in fewer new neurons, while the addition of Fortasyn increased the production of new neurons.</p> Signup and view all the answers

Explain the role of in situ hybridization in the context of brain tissue analysis.

<p>In situ hybridization is used to detect specific mRNAs in tissue sections, allowing for the localization of gene expression in brain tissues.</p> Signup and view all the answers

List the general steps involved in an in situ hybridization protocol.

<p>The steps include incubating with a DIG-labeled probe, incubating with an anti-DIG antibody labeled with alkaline phosphatase, and colorimetric detection using NBT/BCIP.</p> Signup and view all the answers

What is the purpose of using a DIG-labeled probe in in situ hybridization?

<p>The DIG-labeled probe binds to specific mRNAs in the tissue sections, enabling their detection during the hybridization process.</p> Signup and view all the answers

What does the term 'isocaloric control diet' refer to in the context of the study on mice?

<p>An isocaloric control diet provides the same number of calories as the Fortasyn diet but lacks the specific nutrients that Fortasyn contains.</p> Signup and view all the answers

Describe the function of the anti-DIG antibody in the in situ hybridization process.

<p>The anti-DIG antibody binds to the DIG-labeled probe, facilitating subsequent detection and visualization of the target mRNA.</p> Signup and view all the answers

What effect does Fortasyn have on newly-formed neurons after a stroke?

<p>Fortasyn enhances the production of newly-formed neurons compared to the control diet following a stroke.</p> Signup and view all the answers

Flashcards

What is DCX?

A protein marker used to identify newly-formed neurons in the brain.

Ex vivo neuroimaging

The process of preparing tissue samples for visualization and analysis using a variety of techniques.

What is Fortasyn?

An experimental diet that was shown to improve neuron formation after a stroke

What is In situ hybridization?

The technique of detecting specific mRNA molecules within a tissue section.

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Histochemistry

A method of using chemicals to stain cells and tissues to make them visible under a microscope.

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Immunohistochemistry

A technique that uses antibodies labelled with fluorescent dyes to visualize specific proteins in tissue samples.

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What is the first step in the In situ hybridization protocol?

The first step in in situ hybridization involves incubating brain sections with a probe that is labeled with DIG.

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In situ hybridization

A technique used to detect specific RNA molecules in cells and tissues, revealing gene expression.

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What is the second step in the In situ hybridization protocol?

The second step in in situ hybridization involves incubating the tissue sections with an antibody that recognizes DIG and is labeled with alkaline phosphatase.

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What is the third step in the In situ hybridization protocol?

The third step in in situ hybridization involves incubating the tissue sections with a substrate for alkaline phosphatase.

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Whole brain clearing

A technique used to clear tissue samples, making them transparent and allowing for whole-brain imaging.

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What is BCIP?

A chemical used in the final step of in situ hybridization, along with NBT, to produce a blue color where the target mRNA is located.

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iDISCO

A specific type of whole-brain clearing technique that uses a proprietary reagent to make tissue transparent.

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What is tMCAo?

The process of blocking blood flow to a part of the brain, simulating a stroke.

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Sectioning

A technique used to section the brain into thin slices.

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Cryostat

A device that uses a razor-sharp blade to cut extremely thin slices from frozen tissue.

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Primary Antibody Incubation

The process where a primary antibody specifically binds to its target antigen within a tissue sample.

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Blocking

A blocking step is essential in immunohistochemistry to reduce non-specific binding of antibodies to the tissue sample.

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Secondary Antibody

A secondary antibody binds to the primary antibody, amplifying the signal and allowing visualization of the target antigen.

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Enzyme-Labeled Secondary Antibody

Secondary antibodies can be labeled with an enzyme, which allows for visualization through a colorimetric reaction.

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HRP (Horseradish Peroxidase) Enzyme

An enzyme commonly used in immunohistochemistry to detect specific antigens by producing a colored reaction.

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Fluorophore-Labeled Secondary Antibody

Secondary antibodies can be labeled with a fluorescent molecule, allowing for visualization under a fluorescence microscope.

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Alexa Fluor

A common fluorophore used in immunohistochemistry for detecting specific antigens.

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TubeMap

An automated analysis software used in conjunction with iDISCO+ to map and quantify blood vessels in the brain.

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EyeDISCO

A modified iDISCO+ protocol specifically developed for clearing and visualizing entire mouse eyes.

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Dcc (Deleted in Colorectal Cancer)

A protein crucial for the proper guidance of optic nerves during development. A research focus of EyeDISCO studies.

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Cholera toxin β subunit (CTB)

A molecule used in tracer studies to track the paths of neurons, often used in EyeDISCO to visualize neural pathways.

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Tissue Clearing

The process of rendering tissues transparent, allowing for visualization of internal structures.

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Permeabilization

The process of making cells and tissues permeable to antibodies and other probes. This allows for the penetration of antibodies and staining.

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Bleaching

Removing unwanted fluorescent signals from a tissue sample, to improve the clarity and specificity of imaging.

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Fluorescence Microscopy

A type of microscopy that creates images of fluorescently labeled samples. Different colors are used to highlight different structures or components.

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Axon Tracing

A technique for visualizing the trajectories of individual axons throughout the entire central nervous system (CNS).

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Anti-beta amyloid antibodies (PAb #10)

A type of antibody commonly used to detect amyloid-β plaques in brain samples.

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Congo red

A red dye used to stain amyloid-β plaques, often in conjunction with iDISCO+.

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Microglia

A type of immune cell often found surrounding amyloid-β plaques, playing a role in the inflammatory response.

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Iba1

A protein commonly used as a marker for microglia, indicating their presence and activation.

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Volume reconstruction

The process of reconstructing 3D structures from a series of 2D images obtained through microscopy.

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Study Notes

Ex vivo Neuroimaging Tools in Rodents

  • DGCN35: Animal models for brain function and disorders
  • BMS30: Animal models for psychological and neurological disorders
  • Presenter: Bram Geenen, MSc, Research Technician, Department of Imaging, Anatomy, Radboudumc

Overview of Ex Vivo Techniques

  • Techniques used for brain imaging in the lab
  • Goal: Understand steps and choose suitable technique for a research question

Outline of Techniques

  • In vivo experiments, mouse brain
    • Sectioning (cryostat, freezing microtome, paraffin microtome)
    • Whole brain clearing
      • 3DISCO
      • uDISCO
      • iDISCO
    • Histochemistry
      • Immunohistochemistry
      • In situ hybridization

From In Vivo to Ex Vivo

  • Sacrifice animals
  • Anesthesia and analgesia
  • Killing (CO2, cervical dislocation, guillotine, perfusion)
  • Organ harvesting
  • Fixation of organs

Mouse Brain Sectioning

  • Decisions for sectioning or clearing depend on the research question
  • Methods of sectioning
    • Cryostat
    • Freezing microtome
    • Paraffin microtome

Processing Brain Sections

  • Image Acquisition
    • Fluorescent labeling of mice
      • Genetic modification
    • Stain analysis
      • Hematoxylin/eosin
      • Other histochemical stains (e.g., Nissl)

Histochemistry Overview

  • 17th century: Marcello Malpighi studied lung microstructure
  • Larger animals: capillaries and alveoli
  • Insects: Malpighi tubule system
  • 19th century: Camillo Golgi & Santiago Ramón y Cajal

Golgi Staining

  • Developed in 1863
  • Visualizes neurons in brain tissue
  • Steps involve tissue immersion in potassium dichromate and silver nitrate
  • Staining entire neuron, but only a small % of all neurons

Immunohistochemistry

  • Detects specific proteins in tissue sections
  • Antibody binding to antigens
  • Two types of antibodies
    • Polyclonal: injected animal with antigen
    • Monoclonal: fusing B cells to immortal cells
  • Steps
    • Blocking (BSA)
    • Primary antibody incubation
    • Secondary antibody incubation (labeled)
  • Detection methods
    • Colorimetric detection (e.g., HRP, DAB/H2O2)
    • Fluorescent detection (e.g., Alexa Fluor)

Example Study: Multinutrient Intervention After Ischemic Stroke

  • Study in female C57BI/6 mice
  • tMCAO (transient middle cerebral artery occlusion) induced
  • Mice split into control and multinutrient intervention groups
  • Evaluation of cerebral blood flow, structural/functional connectivity, neurogenesis, and function
  • DCX as marker for newly formed neurons

In Situ Hybridization

  • Detects specific mRNA in tissue sections
  • Steps
    • Tissue section incubation with DIG-labeled probe
    • Antibody incubation with alkaline phosphatase enzyme
    • Colorimetric detection with NBT/BCIP substrate
  • Fluorescent detection method is also available

Tissue Clearing

  • Tissue clearing methods
    • Protocol for transparent organs for light-sheet fluorescence microscopy
    • Organic solvent-based
    • Aqueous solvent-based
    • Hydrogel crosslink-based
  • 3DISCO protocol
  • Steps: Dehydrate, dissolve lipids, refractive index matching, image acquisition
  • Methods to clear organs for deeper and broader analysis

uDISCO

  • Optimization of 3DISCO protocol
  • Whole body clearing, used to image larger structures.

iDISCO

  • Protocol for immunolabeling and clearing
  • Visualizes protein expression in 3D
  • Added steps to standard immunohistochemistry
  • Steps: Permeabilization, Bleaching, Blocking, Primary antibody incubation, Secondary antibody incubation, clearing the brain

SHANEL

  • Clearing of intact human organs

Important Concepts in Ex Vivo Neuroimaging

  • Histology, histochemistry, immunohistochemistry
  • Antigens, Antibodies
  • Blocking
  • Primary/secondary antibodies in IHC
  • Colorimetric/fluorescent detection
  • In situ hybridization with DIG/labeled probe
  • Light-sheet microscopy
  • 3DISCO/IDISCO/uDISCO/+ protocols

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Ex Vivo Neuroimaging Tools in Rodents - 2024 PDF

Description

This quiz explores ex vivo neuroimaging tools utilized in rodent studies, detailing techniques for brain imaging, sectioning, and clearing. Aimed at helping researchers understand the processes and methods involved in animal models for investigating brain function and disorders.

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