Protocol
for B-gal staining of tissues with colloidal gold antibodies
- Cut sections on cryostat
- Dry 1 hour at room temperature
- Fix in 100% acetone for 10 minutes at -20oC
- Incubate for 20 minutes at room temperature in 4%
normal goat serum (NGS) in PBST
- Incubate with primary antibody diluted in PBST
for 1-2 hours
- Wash 3 times for 5 minutes with PBST
- Incubate for 1 hour with 4% NGS in PBST
- Wash 3 times for 5 minutes with PBST
- Incubate for 1-2 hours with colloidial gold-antibody
goat-anti-Rabbit diluted in PBST
- Wash 4 times for 5 minutes with PBST
- Wash 2 times for 5 minutes with PBS
- Postfix in 1%-2% glutaraldehyde in PBS with tween-20
for 10-20 minutes
- Wash 3 times for 5 minutes with PBS
- Rinse with distilled water for 10 minutes (several
changes)
Silver enhancement staining
- Place slide in 50/50 mixture of solution B/Water
for 5 minutes
- 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)
- Rinse with distilled water
- Fix in photographic commercial fixative for 2 minutes
- Rinse with distilled water for 5 minutes
- Counterstain with hematoxylin and eosin
- Mount slide
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