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Animal models > Lecture Geenen > Flashcards

Lecture Geenen Flashcards

1
Q

Learning goals

A
  • learn about techniques often used in labs
  • understand their steps
  • be able to advise on which technique to use for a specific research question
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2
Q

In vivo experiments - Mouse brain

A

Either section –>
- Brain sections (Histochemistry, immunohistochemisty, in situ hybridization)
- don’t section –> whole brain clearin (3DISCO, uDISCO, iDISCO

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3
Q

Going from in vivo to ex vivo

A

sacrificing the animals:

  • anesthetics, analgesia
  • killing: Co2, cervical dislocation, guillotine, perfusion
  • Harvesting of organs
  • Fixation of organs
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4
Q

Sections 3 machines

A
  • chryostat (freeze brain to make sections)
  • Freezing microtome (thin sections 4 micrometers)
  • Paraffin microtome
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5
Q

Mouse brain sections

A

Make images
GM mouse with fluorescent label
mouse injected with fluorescent substance

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6
Q

Histochemical staining to visualize cells

A

Usually used - Hematoxylin/eosin
used for BRAIN MORPHOLOGY

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7
Q

Nissl staining (cresyl violet)

A

used for brain morphology, neuron quantification, etc. (stains ribosomes)

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8
Q

History of histochemistry

A

17th century : Marcello Malpighi studied the microstructure of the lungs of different animals

19th century: fathers of neuroscience

  • Camillo Golgi & Santiago Ramon y Cajal

–> very detailed drawings of 2 neurons from cerebellum

Golgi developed Golgi staining to visualize neurons in brain tissue

  • immersion of a block of thick section of tissue in potassium dichromate
  • immersion in silver nitrate
  • deposition of silver chromate
  • section to make thinner sections
  • cover with coverglass
  • investigate using light microscope

–> entire neuron is stained, but only small percentage of all neurons… how does it work? nobody knows

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9
Q

What to do with brain sections?

A

immunohistochemistry:

detecting specific proteins in a tissue section

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10
Q

Immunohistochemistry

A

Antibodies have antigen binding site so that they can bind to a specific antigen

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11
Q

Immunohistochemistry steps of protocol (2 day protocol)

A
  1. Blocking
    incubate with BSA (bovine serum albumin) - (not necessary)
  2. incubation with primary antibody
  3. incubation with labeled secondary antibody
    - labeled with enzyme
    - labeled with a fluorophore
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12
Q

Colorimetric detection

A

secondary antibody labeled with an enzyme
Most used: HRP enzyme and DAB/H2O2 substrate

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13
Q

Fluorescent detection

A

secondary antibody labeled with a fluorophore

most used : Alexa Fluor

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14
Q

Example study multinutrient intervention in stroke research

A

in vivo part (behavioral study)

mouse brain, tissue sections and immunohistochemistry

interest in formation of new neurons in the brain: Marker for doublecortin (DCX) was used

explore how formation of new neurons would be influences by stroke and new nutrients

in situ hybridization

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15
Q

In situ hybridization general steps of protocol

A
  1. Incubation the tissue sections with the DIG-labeled probe
  2. Incubationg with an anti-DIG antibody that is labeled with alkaline phosphatase enzyme
    3 Colorimetric detection by incubating with NBT/BCIP substrate or with fluorescence
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16
Q

Tissue clearing

A

Protocol for making organs (i.e. mouse brain) transparent, to make them suitable for rapid imaging using light-sheet fluorescence microscopy (LSFM)

First publication on clearing: over 100 years ago

Visualizing fluorescently labeled proteins in the brain in 3D, without sectioning the tissue

You see the whole thing at once

17
Q

3 approaches for tissue clearing

A
  • Organic solvent-based
  • Aqueous solvent-based
  • Hydrogel crosslink-based
18
Q

3DISCO

A
  • Dehydrate (tetrahydrofuran - THF - or methanol MeOH)
  • Dissolve lipids (THF or MeOH and dichloromethane - DCM)
  • Refractive index matching in dybenzylether (DBE

Imaging using LSFM, in a glass box filled with the refractive index matching liquid

only take a couple of hours and does not require expensive machines

Transparent mouse brain (only inside liquid) put in the sample holder. The microscope (lightsheetmicroscope) works by shining a sheet of light through the brain. As brain is transparent, the light travels through the brain. Then, fluorescence happens and light is emitted (2D)

objective lens makes 2D picture, but moving it up and down all pictures can be merged creating a 3D picture

19
Q

3 DISCOrefractive index

A

light encounters a medium –> angle and speed at which photons travel changes this angle is called refractive index –> things appear where they should not be

20
Q

uDISCO

A

ultimate three-dimensional imaging of solvent cleared organs

They made an entire mouse transparent in its entirety through the uDISCO

able to see all individual neurons

able to follow a single axon through the entire body

  • Only endogenous fluorescently labeled proteins
21
Q

iDISCO

A

Combine tissue clearing with immunohistochemistry
- visualize protein expression in 3D
- immunolabeling-enabled three-Dimensional imaging of solvent cleared organs
- iDISCO+ : version 2.0 of the protocol

22
Q

iDISCO (In general the same steps as normal immunohistochemistry with extra steps)

A
  • permeabilization
  • bleaching
  • blocking
  • incubation with primary antibody
  • incubation with labeled (fluorescence!) secondary antibody
  • clearing brain (modified 3DISCO method)
    1. Dehydrate using methanol
    2. Dissolve lipids in methanol and dichloromethane
    3. refractive index matching in dibenzylether
23
Q

iDISCO+ method (immunolabeling and clearing) mostly waiting

A

major limitation: endogenous fluorescently labeled proteins (uDISCO) are not preserved

Solution: do immunolabeling on the tags (i.e. anti-GFP antibody

workflow:

  • iDISCO+ method (immunolabeling and clearing)
  • imaging using ultramicroscope (light-sheet fluorescence microscope)
  • use ClearMap scripts to detect cFos staining

finding where cFos was improved

24
Q

wiskers of mice in individual rows

A

innervated to process information

25
Q

Three-Dimensional Study of Alzheimer’s Disease Hallmarks Using the iDISCO Clearing Method

A
  • Application of iDISCO+ to visualize and quantify amyloid-beta plaques in mouse and human brain samples
  • visualization of plaques together with inflammation and vasculature
  • Greater 3D complexity of amyloid-beta plaques in humans, when compared to mice
  • because mice do not get as old as humans
  • permeabilization: getting antibodies inside the tissue
    - detergents and chemicals to break down cell membranes
  • Bleaching: getting rid of autofluorescience
    - hydrogen peroxide and methanol to destroy autofluorescent molecules

Showing beta amyloid, vasculature and lb1

26
Q

Can a mouse get alzheimers disease?

A

No, only humans, you need genetic modification to get some symptoms

27
Q

Mapping the Fine-Scale Organization and Plasticity of the Brain Vasculature

A

iDISCO for entire mouse brain vasculature

  • labeled all the vessels with antibodies

Automatic analysis (TubeMap)

  • brain-wide graph construction and annotation

mouse brain (280 meters of vasculature

Brain wide quantitative analysis

  • vascular organization
    -stroke and disease moled
  • vascular plasticity

Investigate effects of ischemic stroke and sensory loss

28
Q

Revisiting the role of Dcc in visual system development with a novel eye clearing method

A
  • EyeDISCO protocol for whole eye clearing
  • investigate the role of Dcc (guidance molecule) in optic nerve guidance during development
29
Q

Can we clear an entire human organ (entire human brain (8-9 months)

A

no practical application even though fully transparent

30
Q

All the DISCO protocols

A

3DISCO: the original tissue clearing protocol, for transgenic animals (strong endogenous fluorescent labels)

uDISCO: modified 3DISCO protocol, shrinkage used to image larger specimens, for transgenic animals (stronger endogenous fluorescent labels)

iDISCO: using immunohistochemistry (fluorescence) to label proteins, then use 3DISCO protocol to clear tissue

iDISCO+: improved iDISCO protocol, substitute THF for DCM to eliminate (uneven) tissue shrinkage

EyeDISCO: iDISCO+ adapted for eye tissue (de-pigmentation steps)

31
Q

longest part?

A

antibody part takes the longest (have to travel passively through the entire tissue

tissue clearing (couple of hours or over night)

32
Q

Hydrogel crosslink-based: CLARITY

A

Step 1: hydrogel monomer infusion (day 1-3)
Step 2: hydrogel-tissue hybridization (day 3)
Step 3: electrophoretic tissue clearing (days 5-9)

33
Q

Aqueous solvent-based: CUBIC (clear, unobstructed brain imaging cocktails and computational analysis)

A
34
Q

Translational research

A
  • From mouse to man
  • iDISCO of human AD brain tissue (Liebmann paper)
  • Small blocks

Our tries with “fresh” brains
- fixation
- iDISCO + staining
- CSVD
- AD

35
Q

Summary

A
  • Histochemistry
  • Immunohistochemistry
  • In situ hybridization
  • 3DISCO/uDISCO/iDISCO & clearing techniques
36
Q

Important concepts

A
  • Histology, histochemistry, immunohistochemistry
  • Antigens, antibody
  • Blocking, primary antibodies, secondary antibody
  • Colorimetric detection (enzyme), fluorescent detection (fluorophore)
  • In situ hybridization, DIG-labeled probe, target mRNA
  • Light-sheet fluorescence microscope, tissue clearing
  • General steps 3DISCO, iDISCO+
37
Q
A

Animal models (10 decks)

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