Immunolabeling and Transmission Electron Microscopy

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Immunolabeling and Transmission Electron
Microscopy
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Our Samples
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• GFP-Control (GFP 2SC)
• GFP-Treated with Tunicamycin
• Wild Type
• Embryo(PDI2-GFP)

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Goals?
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• GFP protein gene is fused with 2SC gene which
  vacuole targeted gene.
• Treatment with tunicamycin GFP will be folded,
  not glycocylated. Lot of unfolded protein in
  secretory appratus- cause traffic jam. gtGFP
  probably have a hard time to leavegt we expect
  see lot more GFP protein in endomembrane system.

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GFP??
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• Green Fluorescent Protein? Its a protein.
• 238 amino acid sequences.
• From Jelly fish Aquorea
• Victoria Blue light hits
• Green lights out.

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Can be used as protein tracker?
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Fixation
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• Purpose?
• To prepare the structure of cells with minimum
  alteration from the living state
• To protect them against disruption during
  embedding and sectioning
• To prepare specimen (usually tissue) for
  subsequent treatments including staining and
  exposure to the electron beam.
• ? Satisfactory preservation and preparation of
  the cell, in order to see under the electron
  microscope.

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Things need to know about fixing
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• Fixing material or substances are called
  Fixative.
• Ideal fixative Kill tissues quickly?, cause
  minium
  
  shrinkage or swelling.
• -In order to kill quickly
• 1) Freeze drying (e.g our Embryo)
• 2) Chemical fixing (Sample 1,2,3)
• - Speed of penetration
• a) Low molecular weight e.g)
  Formaldehyde
• Molecular weight penetration
  speed? Not
• always!! (e.g HgCl2)

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Things need to know about fixing
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• b) Reacting Radicle
• c) Solubility in lipids
• d) Polarity of molecule
• Separation of liquid phase from solid
  phase of protoplasm is an essential of fixation.
• - Everything is in the water. We changing water
  to fixative solution to organic solvent to
  plastic. Need stable bonds in order to prevent
  breaking chemical bonds, cell components
  translocation or extraction.
• ? Fixatives should give strong chemical bonds
  between
• cell components.

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Fixatives
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• Coagulant Fixatives Flocculate all proteins
  e.g) Ethanol - Considerable change in protein
  structure. Most non additive.
• Non-Coagulant Fixatives Very little
  dissociation of protein from water. Protein
  retain at least some of their reactive group.
  e.g) glutraldehyde, OsO4, acrolein, formaldehyde.
  Most additive.

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Protocol
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• Fix 2 paraformaldehyde 0.1 glutraldehyde in
  0.1M cacodylate with 2mM CaCl2, pH 7.4 approx
  1hr, r.t.
• Wash 0.1M cacodylate with 2mM CaCl2 210min
• Dehydrate 10,30,50,70,85,95,100 ethanol
  5-10min
• Infiltrate 11 ethanol/LR White 1-2hr on rotator
• 12
• 100 LR White overnight on rotator
• 100LR White 1-2 hr on rotator
• Embed in gelatin capsules or in other molds
  where oxygen can be excluded.
• Polymerize with UV light in freezer or in oven
  at 50 C for 12-48 hours.

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Factors affecting quality of fixation
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• 1)pH Normal cells average pH 7.4 everythings
  are stable in this pH.
• 2)Buffer Type types of ion presents affect
  fixation of specimen. Common buffers used for EM-
  Collidine, phosphate, Arsenate, Sodium
  bicarbonate, Veronal acetate, Chromatedichromate.(
  hand out)
• 3)Tonicity(Osmolarity) Rate of penetration
• Iso?, Hypo?, Hyper? Achieved by addition
  of electrolyte(e.g NaCl), Non electrolyte(e.g.
• sucrose) Ca2(prevent swell? And more?)

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Factors affecting quality of fixation
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• 4)Concentration of Fixative
• 5)Temperature and Duration of Fixation.
• -Formaldehyde penetrate rapidly but fix
  slowly
• -Glutaraldegyde, Potassium permanganate
  Penetrate slowly but fix rapidly.

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Chemicals in Fixative
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• -Paraformaldehyde penetrate fast, React with
  many functional groups on proteins including
  amine, thiol, hydroxyl,imidazol, phenolic group.
  Also can crosslink DNA. stabilize protein by
  crosslinking?

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Chemicals in Fixative
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• - Glutaraldehyde Cross link proteins rapidly and
  irreversibly. React with lysine, cysteine,
  histidine, tyrosine, tryptophan.

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Chemicals in Fixative
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• Osmium Tetraoxide (OsO4) Crosslink lipids,
  Reduced osmium can provide more electron
  density-can increase contrast. Most targets are
  unsaturated fatty acid.

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Protocol
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• Fix 2 paraformaldehyde 0.1 glutraldehyde in
  0.1M cacodylate with 2mM CaCl2, pH 7.4 approx
  1hr, r.t.
• Wash 0.1M cacodylate with 2mM CaCl2 210min
• Dehydrate 10,30,50,70,85,95,100 ethanol
  5-10min
• Infiltrate 11 ethanol/LR White 1-2hr on rotator
• 12
• 100 LR White overnight on rotator
• 100LR White 1-2 hr on rotator
• Embed in gelatin capsules or in other molds
  where oxygen can be excluded.
• Polymerize with UV light in freezer or in oven
  at 50 C for 12-48 hours.

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Protocol
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• Fix 2 paraformaldehyde 0.1 glutraldehyde in
  0.1M cacodylate with 2mM CaCl2, pH 7.4 approx
  1hr, r.t.
• Wash 0.1M cacodylate with 2mM CaCl2 210min
• Dehydrate 10,30,50,70,85,95,100 ethanol
  5-10min
• Infiltrate 11 ethanol/LR White 1-2hr on rotator
• 12
• 100 LR White overnight on rotator
• 100LR White 1-2 hr on rotator
• Embed in gelatin capsules or in other molds
  where oxygen can be excluded.
• Polymerize with UV light in freezer or in oven
  at 50 C for 12-48 hours.

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Protocol
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• Fix 2 paraformaldehyde 0.1 glutraldehyde in
  0.1M cacodylate with 2mM CaCl2, pH 7.4 approx
  1hr, r.t.
• Wash 0.1M cacodylate with 2mM CaCl2 210min
• Dehydrate 10,30,50,70,85,95,100 ethanol
  5-10min
• Infiltrate 11 ethanol/LR White 1-2hr on rotator
• 12
• 100 LR White overnight on rotator
• 100LR White 1-2 hr on rotator
• Embed in gelatin capsules or in other molds
  where oxygen can be excluded.
• Polymerize with UV light in freezer or in oven
  at 50 C for 12-48 hours.

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Protocol
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• Fix 2 paraformaldehyde 0.1 glutraldehyde in
  0.1M cacodylate with 2mM CaCl2, pH 7.4 approx
  1hr, r.t.
• Wash 0.1M cacodylate with 2mM CaCl2 210min
• Dehydrate 10,30,50,70,85,95,100 ethanol
  5-10min
• Infiltrate 11 ethanol/LR White 1-2hr on rotator
• 12
• 100 LR White overnight on rotator
• 100LR White 1-2 hr on rotator
• Embed in gelatin capsules or in other molds
  where oxygen can be excluded.
• Polymerize with UV light in freezer or in oven
  at 50 C for 12-48 hours.

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Sectioning
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• Cut with Ultra Microtome.

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Sectioning
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• Thick cut 1micrometer(glass blade)

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Sectioning
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• Thin cut 80nm

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Immunolabeling Protocol
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• Incubate on drops of 5 Na-metaperiodate 40ul
  20min
• Wash with dH2O 40ul 33
• Block with PBST5 milk 40ul 15min
• Incubate with primary antibody 10ul 1hr(110)
• Wash with PBST5 milk 40ul 25min
• Incubate with secondary antibody 10ul
  30min(1100)
• Wash with PBST5 milk 40ul 5min
• Wash with PBS 50ul 25min
• Wash with dH2O 50ul 210min
• Poststain with UrAc and Pb citrate
• Dry and view with TEM

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• Sodium Metaperiodate Stablize polysaccharides
  and glycoprotein. Etching
• Primary Ab Ra anti GFP IgG
• Secondary Ab Goat anti Ra IgG conjugated with
  colloidal gold.

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Colloidal gold
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• Gold particle, not have defined structure,found
  in suspension in water, negatively charged.
• Liquid usually appear red color(particles less
  than 100nm), conjugated with monoclonal
  antibodys Fc region by ionic interaction which
  lysine, tryptophan, and cysteine are found
• Available by size.
• We used 10nm.

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Transmission Electron Microscopy
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Grid 2 Treated with tunicamycin
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More grid2
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Grid 4 Embryo
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Poststain??
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• UrAc and Pb citrate heavy metal? Electron
  scattering power?
• Make a circle!! (penetration or not??)

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• Unstained
  Stained

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Discussion
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• We didnt even look at grid 1 and 3.
• We couldnt find cluster of gold particle on our
  sample 2.
• We know GFP is in there by tracking with
  epi-fluorescence microscopy and western blot.
• Conclusion
• ?Something wrong!!!

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So, What can we do? Huh?
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• It could be fixation problem.
• Maybe we washed too wrong.
• Maybe they are too shy.