Cresyl Violet staining

Perform this procedure under a chemical fume hood. Wear two layers of nitrile gloves. Lab coat and eye protection required.

Hazardous chemicals used in this procedure: acetic acid, xylene, DPX

1. In 0.9% NaCl (aq), spread out vibratome sections of the perfusion-fixed brain in sequence from rostoral to caudal (refer to the rat brain atlas).
2. Mount the sections in sequence onto glass microscope slides.
3. Air dry the mounted sections 2-3 days.
4. Load staining rack with slides with the sections.
5. Prepare staining dishes: purified water, Cresyl Violet, 100% ethanol, 100% ethanol with 0.1% acetic acid (glacial), 100% ethanol × 4, xylene × 3
6. Place the rack containing slides in purified water, 2 min
7. 100% ethanol × 2, 3 min each
8. Xylene, 4 min
9. 100% ethanol × 2, 4 min each
10. 95% ethanol, 4 min
11. 75% ethanol, 4 min
12. Purified water × 2, 1 min each
13. Drain and briefly dry the sections
14. cresyl violet solution, 2 min
15. Rise briefly in 100% ethanol
16. 100% ethanol with 0.1% acetic acid (glacial), ~1-2 min
    1. 200 ml working vol EtOH, 200 µl Acetic Acid                                                     
17. 100% ethanol × 4, 2 min each (check staining with a microscope after 2 changes)
18. Xylene × 3, 3 min each
19. Coverslip with DPX mountant - keep slides in xylene until coverslip is applied
20. List of slides:
21. Let dry in hood ~48 hrs