Immunolabeling of Cells and Transmission Electron Microscopy

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Immunolabeling of Cells and Transmission Electron
Microscopy

• Ike Miguel
• June 30, 2006
• ABE Workshop 2006

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Purpose

• Using TEM to label specific sites in Arabidopsis
  thaliana.
• Goal
• Overview of Immunolabeling
• Preparation of samples and viewing
• Observations of results
• Thoughts

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Antibodies are used in immunolabeling of cells

• Immunoglobulins, or antibodies, detect foreign
  bodies and attach at specific sites called
  epitopes
• Antibodies fused to myeloma cells where they are
  cloned indefinitely and screened
• in vitro and in vivo
• Indirect method used to our purposes

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Antibody Structure

• 4 polypeptide chains
• Two light, VL and CL
• 2 heavy chains determine fuctional activity in
  vivo
• IgG, IgA, IgM, IgE, IgD

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Use of Antibodies for Immunolabeling

• High level of specificity needed due to
  non-covalent, weak bonding
• Eliminate non-specific binding
• Optimal conditions include reagants, pH, heat.
• Controls are very important!
• Negative controls to discriminate non-specific
  binding

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Tranmission Electron Microscopy

• Zeiss 10/A conventional TEM
• Easy operation
• Uses film for images

• LEO 912 Energy-Filtering TEM
• Little as 0.5um sections
• EELS, ESI
• Digital images
• Ultramicrotomy, 60-90nm sectioning in resin

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Immunolabeling with Gold by Indirect Method

• Gold small, round and dense
• Easily detectable
• 110 and 1100 colliodal gold-labeled conjugated
  to primary antibody

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Preparation of Specimens by Embedding

• Preparation is limiting factor!
• Chemical fixation with aldehydes which
  cross-links proteins
• Paraformaldehyde for grids
• Dehydrate
• Infiltrate with resin
• Polymerize
• May stain prior to polymerization
• 7 10 days to fix. May need to repeat.

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Ultramicrotomy and transfer to grids

• Glass knife can cut ucron thick sections
• Diamond knife can cut as small as 2-3um
• Our samples 80nm 1um thick
• 60-80nm max thickness for transmission of light
• 2 sections embedded per grid
• Avg of 6 sections per grid (2 20)

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Post-Embedding and Labeling of Specimens

• Dilution series and controls!
• Sectioned on Formvar coated grids
• Pretreat and etch with Na-metaperiodate/
  PBS-glycine
• Wash with TBST (detergent, ampiphatic, nonionic)
• Block with milk (lipid, naturally sticky)
• 1o antibody
• Wash with TBST
• 2o antibody, colliodal-gold
• Wash with TBS and H20 to rinse any Ppt

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TEM LEO912 Energy-Filtering TEM

• Tungsten cathode used to produce electron
  illumination source
• Nitrogen for carbon residue
• Accelerated through column
• EMF used to bend and focus illumination
• Grid sample on objective lense
• Projector lense produces visible light
• Beam strikes CCD strip for histogram results

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GFP, DTT treated

• Grid 1 No pretreatment, 110 anti-GFP (1o-Ab),
  110 gold (2o-Ab)
• DTT dithiothritol used to denature GFP
• Expected labeling on ER, vacuole, or vesicles.
• What is this?

Fig. 1 Numerous binding to organelle. Group 1.
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GFP DTT non-specific binding

• Grid 2 Glycine, 110 anti-GFP (1o-Ab), 110 gold
  (2o-Ab) labeling
• Non-specific binding is expected result
• Thoughts?
• Vascular system or chloroplast

Fig. 2 Non-specific binding
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Group 2 GFP no DTT treatment
Grid 3. No pretreatment, 110 anti-GFP, 1100
gold. Possible labeling on vacuole.
Grid 4. Na-Periodate, 1100 anti-GFP, 1100 gold.
Random labeling
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PDI2

• Expected labeling on starch granules
• No pretreatment
• At high concentrations primary antibody may have
  a large number of non-specific binding.

Grid 1. No pretreatment, 110 anti-PDI-2, 110
gold. Observing non-specific binding. Group 3
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Group 3 PDI2
Grid 1. A lot of non-specific binding but closer
observations may give insight.
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Group 4 PDI2
Grid 4. Na-Periodate, 110 anti-PDI2, 11000?
gold. Labeling of starch granules
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Group 4 Control
Grid 4. Control with no primary antibody.
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Group 4 CNGC

• Cyclic nucleotide gated channel
• Allows passage of K
• Expected labeling on plasma membrane

Grid 2. Glycine, 110 anti-CNGC, 110 gold
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Group 4 CNGC
Grid 2. Gycine, 110 anti-CNGC, 110 gold.
Possible communication.
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CNGC
Grid 3. No pretreatment, 110 anti-CNGC, 1100
gold. Group 4
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Errors and Conclusion

• Preparation is the limiting factor!
• Gold labeling antibody may have been prepared in
  the wrong concentrations.
• A set of new dilutions may be needed.