- Proteins and Peptides
- Lysates and Cell Lines
Immunostaining of frozen sections using streptavidin or Exvtravidin peroxidase and DAB
1. Make 10 micron frozen sections.
2. Fix tissue sections on slides in cold acetone, 10% buffered folrmalin or 4% paraformaldehyde for 8 to 10 min.
3. Rinse slides with PBS by immersion for 2 min. Repeat.
4. Block all slides with 5% milk in PBST for 30 min. at RT.
5. Remove blocking solution and add NB 110-58771 LRRK2 primary antibody 1:100 to 1:200 in PBS with 0.1% triton X-100 with 5% milk.
6. Incubate slides at 4degrees Celcius, overnight.
7. Rinse slides with PBST by immersion for 2 min. Repeat.
8. Add biotinylated donkey anti-rabbit secondary antibody (1:500 in PBST with 5% milk). Incubate 1 hour at RT.
9. Rinse with PBST by immersion 2 min. Repeat.
10. Add streptavidin-HRP or Extravidin-HRP. Incubate at RT for 1 hr.
11. Rinse with PBS by immersion for 1 min. Repeat.
12. Make DAB solution.
13. Add DAB to slides. Incubate 5-7 minutes.
14. Stop DAB reaction by immersion in water.
15. Counterstain sections, if desired.
16. Dehydrate,defat and coverslip slides.