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Comparative Physiology @ Guelph

Fluorescent Staining Protocol for Mucin Vesicles

  • Dissolve 2 mg of fluorescent dye (Flourescein 5 isothiocyanate) in 200 ul of solvent (dimethyl sulphoxide).
  • Add 20 ul of dye solution to 1ml of mucin vesicles in stabilization buffer.
  • Mucin vesicles should be allowed to incubate in dye for 1 hour.
  • Centrifuge dyed vesicles 5 times at 4000rpm for 4 minutes.
  • After each centrifuge, pipette off the top layer of liquid to remove excess dye then flush vesicles with stabilization buffer.
  • After the last centrifuge, remove the top layer of liquid, but do not flush with more stabilization buffer in order to yield a more concentrated sample.