Imaging the Nervous System Flashcards
Why do we visualise cells of the nervous system? (3)
- Cell Morphology
- Cell Projections and Connections
- Protein Expression
How does a fluorescent microscope work? (5)
- Excitationlight is shoneonto thesample
- Excitation filterdetermineswavelength of excitationlight
- Excitation light isabsorbedby the fluorescent tag
- Electronsareexcited
- Emissionlight isreleasedas the electrons relax
How does a confocal microscope work? (6)
- Excitationlight from a laser is shoneonto thesample
- Excitation pinholecreates a verynarrow beam of the excitation lightonto the sample
- Excitation light isabsorbedby thefluorescent tag in particularplane offield
- Electronsareexcited
- Emissionlight isreleasedas theelectrons relax
- Emission filteris selective for the lightemitted from thespecific plane
What does SEM allow us to see? And Why?
- 3D image of surface
- Electronsbounceof the surface
What does TEM allow us to see? And why?
- Internal structure
- Electronspass throughsample
How is tissue prepared for viewing? (6)
- Tissue is harvested and preserved by being snap freezing (straight into liquid nitrogen, prevents ice crystals) or chemically fixed e.g. PFA.
- Tissue is cut whilst frozen (easily) or if chemically fixed it is then embedded in wax or resin. A Microtome or cryostat is used to cut the tissue. µm thick.
- Tissue is mounted onto a glass slide (Snap frozen tissue is chemically fixed e.g. PFA)
- Staining
- Coverslip
- Image
What colour is Hematoxylin?
Dark blue/Violet
What charge does Hematoxylin have?
Positively charged/basic
What does Hematoxylin bind to?
- Binds tonegativelychargedDNAandRNAin thenucleus
- Binds to negatively chargedRNAin theroughendoplasmic reticulum(RER)
What colour is Eosin?
Pink
What charge does Eosin have?
Negatively charged/acidic
What does Eosin bind to?
Binds topositivelychargedamino acid sidechainsin thecytoplasm
What are the other names for Nissl Stain?
aniline, thionine, orcresylviolet
What colour is Nissl Stain?
Dark blue/Violet
What charge does Nissl Stain have?
Positively charged/basic
What does Nissl Stain bind to?
- Binds tonegativelychargedDNAandRNAin thenucleus
- Binds to negatively chargedRNAin therough endoplasmic reticulum(RER)
What colour is Golgi Stain?
Black
How does the Golgi Stain stain tissue?
Impregnates tissue withpotassium dichromate andsilver nitrate
What colour is Osmic Acid?
Black
What does Osmic Acid bind to?
Binds to lipids, myelin is a fatty sheath.
What is another use of Osmic Acid?
A fixative for tissue in Electron Microscopy.
What is immunohistochemistry?
Uses Antibodies which are fluorescent as it can be tagged with an enzyme to form an enzyme complex which can produce a dye precipitate in the presence of its substrate as it binds to the protein\antigen of interest. Therefore antibodies can be used as cell markers and to view protein expression.
How are antibodies produced for immunohistochemistry?
- Antibody raised against antigenofinterest
- Antigen introducedto animal
- Stimulatesimmune responseandantibody production
- Collectand purify antibodies
What is direct immunohistochemistry?
Single antibody binds directly to the antigen and are tagged with fluorophore or an enzyme complex.
What is Indirect immunohistochemistry?
Signal amplification.
Uses two antibodies - a primary and secondary.
- The primary antibody binds directly to the antigen.
- The secondary antibody binds to the primary antibody and is tagged with fluorophore or an enzyme complex
How is the antigen named?
Name of antigen e.g., GFAP
How is the primary antigen named?
Animal that produced it anti-antigen E.g., Rabbit anti-GFAP.
How is the secondary antigen named?
Animal that produced it anti- animal that produced primary antibodies e.g., Donkey anti-rabbit.
