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.


  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.


  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