Lecture Geenen

Question 1

Learning goals

Answer 1

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

Question 2

In vivo experiments - Mouse brain

Answer 2

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

Question 3

Going from in vivo to ex vivo

Answer 3

sacrificing the animals:

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

Question 4

Sections 3 machines

Answer 4

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

Question 5

Mouse brain sections

Answer 5

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

Question 6

Histochemical staining to visualize cells

Answer 6

Usually used - Hematoxylin/eosin
used for BRAIN MORPHOLOGY

Question 7

Nissl staining (cresyl violet)

Answer 7

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

Question 8

History of histochemistry

Answer 8

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

Question 9

What to do with brain sections?

Answer 9

immunohistochemistry:

detecting specific proteins in a tissue section

Question 10

Immunohistochemistry

Answer 10

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

Question 11

Immunohistochemistry steps of protocol (2 day protocol)

Answer 11

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

Question 12

Colorimetric detection

Answer 12

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

Question 13

Fluorescent detection

Answer 13

secondary antibody labeled with a fluorophore

most used : Alexa Fluor

Question 14

Example study multinutrient intervention in stroke research

Answer 14

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

Question 15

In situ hybridization general steps of protocol

Answer 15

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

Question 16

Tissue clearing

Answer 16

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

Question 17

3 approaches for tissue clearing

Answer 17

• Organic solvent-based
• Aqueous solvent-based
• Hydrogel crosslink-based

Question 18

3DISCO

Answer 18

• 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

Question 19

3 DISCOrefractive index

Answer 19

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

Question 20

uDISCO

Answer 20

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

Question 21

iDISCO

Answer 21

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

Question 22

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

Answer 22

• 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

Question 23

iDISCO+ method (immunolabeling and clearing) mostly waiting

Answer 23

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

Question 24

wiskers of mice in individual rows

Answer 24

innervated to process information

Question 25

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

Answer 25

- 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

Question 26

Can a mouse get alzheimers disease?

Answer 26

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

Question 27

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

Answer 27

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

Question 28

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

Answer 28

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

Question 29

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

Answer 29

no practical application even though fully transparent

Question 30

All the DISCO protocols

Answer 30

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)

Question 31

longest part?

Answer 31

antibody part takes the longest (have to travel passively through the entire tissue tissue clearing (couple of hours or over night)

Question 32

Hydrogel crosslink-based: CLARITY

Answer 32

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

Question 33

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

Question 34

Translational research

Answer 34

- From mouse to man - iDISCO of human AD brain tissue (Liebmann paper) - Small blocks Our tries with "fresh" brains - fixation - iDISCO + staining - CSVD - AD

Question 35

Summary

Answer 35

- Histochemistry - Immunohistochemistry - In situ hybridization - 3DISCO/uDISCO/iDISCO & clearing techniques

Question 36

Important concepts

Answer 36

- 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+