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HOME > Protocols > Histology > Staining protocols > Protocol for B-gal staining of tissues with colloidal gold antibodies

Protocol for B-gal staining of tissues with colloidal gold antibodies

  1. Cut sections on cryostat
  2. Dry 1 hour at room temperature
  3. Fix in 100% acetone for 10 minutes at -20oC
  4. Incubate for 20 minutes at room temperature in 4% normal goat serum (NGS) in PBST
  5. Incubate with primary antibody diluted in PBST for 1-2 hours
  6. Wash 3 times for 5 minutes with PBST
  7. Incubate for 1 hour with 4% NGS in PBST
  8. Wash 3 times for 5 minutes with PBST
  9. Incubate for 1-2 hours with colloidial gold-antibody goat-anti-Rabbit diluted in PBST
  10. Wash 4 times for 5 minutes with PBST
  11. Wash 2 times for 5 minutes with PBS
  12. Postfix in 1%-2% glutaraldehyde in PBS with tween-20 for 10-20 minutes
  13. Wash 3 times for 5 minutes with PBS
  14. Rinse with distilled water for 10 minutes (several changes)

Silver enhancement staining

  1. Place slide in 50/50 mixture of solution B/Water for 5 minutes
  2. Transfer the slide to the developer 50/50 solution A/B (mixed just before use) for 4-18 minutes in the dark at room temperature (time depends on concentration of target)
  3. Rinse with distilled water
  4. Fix in photographic commercial fixative for 2 minutes
  5. Rinse with distilled water for 5 minutes
  6. Counterstain with hematoxylin and eosin
  7. Mount slide

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