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Low Fade Mounting Medium

For mounting fluorescently labelled specimens:

Reagents:
  • Polyvinyl alcohol (Aldrich 36,062-7 or Sigma P8136-7) low MW cold water soluble (possible carcinogen)
  • Trizma Base (Sigma T1503)
  • NaH2PO4
  • 1% Phenol Red
  • Glycerol
  • Chlorobutanol (1,1,1 tri chloro 2 methyl 2 propanol, Sigma T5138).
To make Tris-PO4 Buffer:
  • 1M Trizma Base (12.11%) 12.11g/100mls
  • 1M NaH2PO4.2H2O (15.62%) 15.62g/100mls
  • Titrate 50 ml of Trizma with the PO4 dropwise to give pH 9.0.
Method:
  1. Mix 5ml Tris-PO4 buffer and 75ml dH2O in Ehrlinmeyer flask.
  2. Add 2-3 drops of 1% phenol red
  3. Add 20g PVA to solution, invert a beaker over the flask neck and place in 60oC oven. Swirl every now and then to dissolve. It takes 2-3 hours.
  4. Slowly add 30ml glycerol.
  5. Add 100mg chlorobutanol. pH should not exceed 8.2, adjust with Tris- PO4.
  6. Store in syringes at -20oC and the working aliquot at 4oC.

Comments:
Do not stir rapidly as bubbles form.
FITC fluorescence is best at pH 8.5 - 9.
I am unsure if this adds any benefit to the AlexaFluor dyes.
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