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 -20^oC
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

15. Place slide in 50/50 mixture of solution B/Water for 5 minutes
16. 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)
17. Rinse with distilled water
18. Fix in photographic commercial fixative for 2 minutes
19. Rinse with distilled water for 5 minutes
20. Counterstain with hematoxylin and eosin
21. Mount slide