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scientificprotocols/protocol.md

Last active August 29, 2015 14:03
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Beta-Glactasidase Stain
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protocol.md

Solutions

  1. Solution A
  • 5mM potassium ferricyanide, crystalline
  • 5mM potassium ferrocyanide, trihydrate
  • X-gal Stock Solution
    • 40mg/mL in DMSO (100mg in 2.5ml DMSO)
  • Final X-gal Solution
    • Dilute X-gal Stock Solution 1:30 in Solution A
    • (warm Solution A to 37°C to prevent precipitation of X-gal)

Staining Protocol

Start with fixed frozen 10μm tissue sections on pap-penned slides

  1. Allow slides to completely dry for 1 hour
  • Fix tissue with 2% formaldehyde + 0.2% glutaraldehyde for 5 min.
  • Rinse with 1xPBS
  • Incubate slides in x-gal solution in humidity chamber at 37°C overnight
  • Rinse with 1xPBS
  • Immerse slides in 100% ethanol until all precipitate dissolves (1-3 hours)
  • Place slides in:
    • a. Eosin Y 25 seconds
    • b. 95% Ethanol 1 minute
    • c. 100% Ethanol 1 minute
    • d. 100% Ethanol 1 minute
    • e. 100% Ethanol 1 minute
    • f. Xylene 2 minutes
    • g. Xylene 10 dips
  • Coverslip slides using Cytoseal 60

DOI

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