Alcian Blue Staining

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  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