Research Technologies Branch

Biological Imaging Section

Protocols: Mowiol Preparation

Preparation of Mowiol Coverslip Mounting Solution for Fluorescence Microscopy

This protocol describes the preparation of a coverslip mounting solution called Mowiol. Mowiol (Mowiol 4.88, Calbiochem catalog number 475904) is a solution of polyvinyl alcohol. This solution normally hardens overnight after slide preparation, and does not require the coverslips to be sealed with nail polish. The addition of PPD (see below) is recommended to reduce bleaching of fluorescent probes.

1. Put 6 g glycerol in a 50 ml plastic centrifuge tube and add a small stir bar.
2. Add 2.4 g Mowiol (Calbiochem); stir to mix.
3. While stirring, add 6 ml distilled water and leave 2 hours at room temperature.
4. Add 12 ml .2M Tris (pH 8.5).
5. Add a small amount of NaN3 (Sodium Azide) such that the final concentration is .02% (optional).
6. Incubate the tube in hot water (50 – 60ºC) for 10 minutes to dissolve the Mowiol. This can be repeated over several hours if necessary.
7. Centrifuge at 5000 g for 15 minutes to remove any undissolved solids. Store 1ml aliquots in eppendorf tubes at -20ºC.
8. Warm tubes to room temperature for use. Opened tubes can be stored at 4ºC for approximately 1 month. Discard if any crystalline material is seen in the tube or on the slides.
9. Leave coverslipped slides in the dark overnight to harden before oil immersion lenses are used.

Note: Do not use so much mounting solution that the coverslips are floating. Normally, 15 – 20 ul is sufficient for a 22 x 22 mm coverslip. 22 x 50 mm coverslips require about 40 – 50 ul.

Addition of PPD

PPD = p-phenylenediamine (1,4-Benzenediamine hydrochloride, Sigma # P1519)

1. Make up a 0.1% aqueous solution of PPD
2. Aliquot and freeze in 1 ml eppendorf tubes wrapped in aluminum foil
3. To use, thaw and add 1 part ppd solution to 9 parts Mowiol
4. Refreeze immediately after use
5. Discard any solution that becomes discolored (pink/brown)

Note: This compound is carcinogenic and should be handled with care.

Reference: Vanes K, Brandt P. Retardation of immunofluorescence during microscopy. J. Histochem. Cythochem. 1985. 33:755-61.

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