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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