Cresyl Violet Acetate Stain

Purpose

Ø to examine neuronal loss and accompanying gliosis

Procedure

Prep

Ø Tissue is mounted onto slides after they have been rinsed in PB and allowed to dry

Ø Slides are then placed into slide holders back to back (20), ready to be stained

Ø Make CV solution Under Fume Hood with Gloves, Eye and Mask

a) Mix 4.0 g sodium acetate in 30 ml dH₂O

b) Mix 9.72 ml GAA (Glacial acetic acid) with 170 ml dH₂O

c) Mix 2.5 g Cresyl Violet Acetate with 300 ml dH₂O

q Combine A + B + C and mix well

q Filter through Whatmann Filter #1 with Buchner Funnel

q pH to 3.7

Ø Set up staining solutions in sequence following the procedure below, apply tray handle and have a stop watch close by – Wear Gloves

Staining

1. 100% Alcohol 1 min
2. Chloroform 5 min
3. 100% Alcohol 1 min
4. 95% Alcohol 1 min
5. 70% Alcohol 1 min
6. dH₂O 30 sec
7. CV solution 4-10 min (varies)
8. dH₂O 15 sec
9. dH₂O 15 sec
10. dH₂O 15 sec
11. 70% Alcohol 1 min
12. 95% Alcohol 1 min
13. 95% Alcohol + GAA 1.3 min
14. 95% Alcohol 1 min
15. 100% Alcohol 1 min
16. Xylene 1 min
17. Xylene 1 min
18. Xylene 5-20 min
19. Remove 2 slides from rack and clean with Kim Wipe – apply 3
20. drops of Cyotoseal and cover slip. Continue until complete.