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Etching of the plastic

Good results have been reported when doing immuno electron microscopy on Epon-Araldite embedded tissue by etching the plastic with sodium metaperiodate using the method described in "Ultrastructural Localization of Antigenic Sites on Osmium-Fixed Tissues Applying the Protein A-Gold Technique" by M. Bendayan and M. Zollinger, Journal of Histochemistry and Cytochemistry, Vol 31:1, (101-109), 1983.

Negative Staining

A powerful technique can be the use of immunolabeling procedures followed by negative staining on virus particles.

See: Spehner, D., R. Drillien, F. Proamer, C. Houssais-Pecheur, M. A. Zanta, M. Geist, K.Dott, and J. M. Balloul, Virology 273:9-15 (2000).

Osmium Coating for BSE and Cell Surface Tagging

The use of colloidal gold reagents for the surface tagging of cells has been reported by many researchers. However, it is necessary to make the sample conductive and the long time accepted procedure for doing this has been by carbon coating. However, a carbon coated sample lacks the three dimensional "appearance" of a gold coated sample, for example, thereby making the results a challenge to properly interpret.

Gold or any other precious metal, applied by sputtering techniques, would always result in a layer that is just too thick to all the gold signal to pass through to the BSE detector. However, when osmium metal is deposited with the OPC Osmium Plasma Coater, a layer so much thinner can be applied and still achieve the desired conductivity, that its presence does not interfere at all with the detection of the gold tagged cells.

Pre-embedding Labeling of Cell Surface Antigens for EM

We present these protocols in good faith to our customers. They have been validated when using SPI-Mark™ Colloidal Gold Reagents, however, we would be reluctant to extrapolate the results one might get if using reagents of different brands.

Protocol:

  1. Isolate cells from tissue with trypsinization or separate blood cells by gradient centrifugation.

  2. Prefix cells in 0.1% SPI-Chem™ EM Grade Gluteraldehyde or 2-4% SPI-Chem Paraformaldehyde in PBS depending on antigen sensitivity.

  3. Wash 3 times in PBS containing 50mm glycine to quench aldehydes.

  4. Block cells for 30 mm in PBS + 1% BSA + 10% normal serum (second antibody species) + 0.1% sodium azide, pH 7.4

  5. Wash twice in PBS + 1% BSA

  6. Incubate with primary antibody in incubating buffer in PBS + 1% BSA + 0.1% Tween 20 + 1% normal serum (second antibody species) for 1 hour.

  7. Wash twice in incubating buffer

  8. Incubate with gold-labelled second antibody in incubating buffer (step 6) for 1 hour.

  9. Wash twice in PBS

  10. Fix in 1% gluteraldehyde in PBS for 15 mm

  11. Wash twice in distilled or deionized water.

Optional silver enhancement:

  1. Silver enhance for 1-10 minutes to enlarge gold particles using the SPI-Chem Silver Enhancement Kit

  2. Wash twice in distilled water to stop enhancement and remove silver solutions.

Embedding:

  1. Spin to a pellet and dehydrate in alcohol

  2. Embed in epoxy, using SPI-Chem SPI-Pon™ 812 or an acrylic resin system, such as LR White™ or Unicryl™ or Lowicryl™ "original" or Lowicryl Monostep.

  3. Section and stain for EM or LM

References

  • C Ferrari, et al (1989) '~Pre-embedding immunogold staining of cell surface antigens performed on suspended cells and tissue sections" in Hayat MA, Colloidal Gold, Vol. 2 (Ch 16)