SPI-Mark™ Colloidal Gold Reagents: Protocols

Chinese

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:

12. Silver enhance for 1-10 minutes to enlarge gold particles using the SPI-Chem Silver Enhancement Kit
13. Wash twice in distilled water to stop enhancement and remove silver solutions.


Embedding:

14. Spin to a pellet and dehydrate in alcohol
15. 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.
16. Section and stain for EM or LM

1. 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)

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