Alcian Blue Staining

1. Fix sample O/N in 4% PFA freshly made
2. Wash in PBS, 3X5mins, 3X30mins

3. Wash 20mins in PBST

4. Equilibrate in EtOH, 25, 50%, 10 mins each

5. Place in Alcian blue staining solution: 9mg alcian blue dissolved in 60mls EtOH plus 40mls glacial acetic acid , make fresh every time.

6. Staining is carried out at 37C, can take anytime between 1 hr to 2days. Check every so often as the solution will precipitate.

7. When staining is complete wash with EtOH/acetic acid mix for 1hr at RT

8. Rehydate gradually, 90, 70, 50 25% EtOH/PBS.

9. Then go through glycerol series 25, 50, 75% glycerol/PBS