Preparation and Manipulation of Cells and Samples for Microscopy

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Preparation and Manipulation of Cells and Samples
for Microscopy
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Preparation and Manipulation of Cells For
Microscopy

• Fixation and fluorescent labeling of cells
• Expression of wild-type and mutant DNA
• Introduction of proteins into cells.
• Use of caged probes to manipulate small molecules
  in cells.

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Preparation and Manipulation of Cells For
Microscopy

• Fixation and fluorescent labeling of cells
• Expression of wild-type and mutant DNA
• Introduction of proteins into cells.
• Use of caged probes to manipulate small molecules
  in cells.

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Importance of Fixation for Microscopy
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Why Fix Samples?

• Spatial location of proteins can provide
  important information about a particular
  proteins function.
• Fixation freezes proteins and DNA in place.
• Preserves samples and protects against autolysis.

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Fixation Methods

• Methanol/Ethanol/Acetic acid-coagulative
  fixatives.
• Crosslinking fixatives
• Formaldehyde
• Glutaraldehyde

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Formaldehyde Fixation
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Formaldehyde Fixation-Caveats

• Relatively slow fixative. Usually takes 24-48
  hours for complete fixation.
• Large osmotic gradients. May cause varying
  results in different tissue compartments.
• Can destroy antigens.

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Glutaraldehyde Fixation
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Glutaraldehyde Fixation
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Glutaradehyde Fixation-Caveats

• Rapid fixative
• Extensively crosslinks proteins
• More likely to destroy antigens.
• Preserves three dimensional structure best.
• Autofluorescence is enhanced. Can be a problem.

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Preparation and Manipulation of Cells For
Microscopy
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Preparation and Manipulation of Cells For
Microscopy

• Fixation and fluorescent labeling of cells
• Expression of wild-type and mutant DNA
• Introduction of proteins into cells.
• Use of caged probes to manipulate small molecules
  in cells.

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Introduction of DNA Into Cells
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Cationic Lipids
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Insertion of DNA/Protein-Proteo-lysosomes
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Gene Delivery-Microinjection
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Gene Delivery-Microinjection

• Technically difficult
• Relatively small numbers of cells can reasonable
  be injected.
• Damages the cells.
• Can be used to deliver proteins, drugs or DNA.

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Gene Delivery-Microinjection
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Gene Delivery-Adenovirus/ AAV/Lentivirus/Retroviru
s
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Gene Delivery-Adenovirus/ AAV/Lentivirus/Retroviru
s

• Advantages
• High probability of gene expression
• Can do biochemistry on cells to confirm light
  microscopy findings.
• Can express more than one gene in a particular
  cell.
• Disadvantages
• Cytopathic changes can occur-changes the biology.
• Cannot be used for stable expression

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Preparation and Manipulation of Cells For
Microscopy

• Fixation and fluorescent labeling of cells
• Expression of wild-type and mutant DNA
• Introduction of proteins into cells.
• Use of caged probes to manipulate small molecules
  in cells.

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Use of Caged Compounds To Study Cellular Function
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Caged Compounds

• Nucleotides
• ATP
• ADP
• cAMP
• cGMP
• GTP
• GDP

• Neurotransmitters
• Nitric oxide
• Aspartic acid
• Glutamic acid
• Inositol 1,4,5-triphosphate
• g-Aminobutyric acid

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Caged Compounds

• Calcium Regulator
• NP-EGTA
• DM-Nitrophen

• Caging Reagents
• 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene
• 2-nitroveratryloxychoro-carbamate

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Caged Compounds-Characteristics

• Caged reagent should react with the protein of
  interest in aqueous solution in physiologic
  relevant conditions
• The bond between the caged compound and the
  protein should be stable at physiologic
  conditions
• The caged group should exhibit high molar
  absorptivity (gt1000 M-1 cm-1)
• Photoisomerization reactions leading to bond
  cleavage should proceed with a reasonable quantum
  yield (gt0.1) and exhibit an action spectrum in
  the near-UV wavelength region (340-400 nm) to
  avoid interference with other biomolecules.
• The photoproducts of photoactivation should not
  react with other functional groups in the protein.

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Preparation and Manipulation of Cells For
Microscopy

• Additional considerations
• Time dynamics of the process you are studying.
• Time/temperature/pH/ionic composition/concentratio
  n
• Keep samples happy
• Can you get an answer by microscopy alone?