Thursday 1 September 2011

Preparation Of Mowiol Mounting Medium

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. Put 6 g glycerol in a 50 ml plastic centrifuge tube and add a small stir bar.
1. Add 2.4 g Mowiol (Calbiochem); stir to mix.
2. While stirring, add 6 ml distilled water and leave 2 hours at room temperature.
3. Add 12 ml 0.2M Tris (pH 8.5).
4. Add a small amount of NaN3 (Sodium Azide) such that the final concentration is 0.02% (optional).
5. Incubate the tube in hot water (50 – 60ºC) for 10 minutes to dissolve the Mowiol. This can be repeated over several hours if necessary.
6. Centrifuge at 5000 g for 15 minutes to remove any undissolved solids. Store 1ml aliquots in eppendorf tubes at -20ºC.
7. 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.
8. Leave cover slipped slides in the dark overnight to harden before oil immersion lenses are used or put at 37oC for about 10 min.
Note: Do not use so much mounting solution that the coverslips are floating. Normally, ~5 µl for a 10mm round coverslip, 15 – 20 µl is sufficient for a 22 x 22 mm coverslip. 22 x 50 mm coverslips require about 40 – 50 µl.

Benchtop Preparation for TEM


Transmission electron microscopy (TEM) can be a fantastic way to obtain a wealth of information about biological structures and processes. Unfortunately the conditions inside a TEM are not particularly hospitable for biological tissues. To successfully pass electrons through TEM specimens, they must be thinner than a wavelength of visible light, resistant to hard vacuum and completely dehydrated. (In other words: dead!)

Preparing biological specimens for TEM therefore requires preservation, dehydration and embedding of the sample for sectioning on an ultramicrotome. Specimen preparation is arguably the most important factor in acquiring high quality images, so here is a generic (“benchtop”) protocol that we use as a guide when preparing samples for ultra-thin sectioning. See below the protocol for important notes... but first...

DISCLAIMER: This protocol is meant strictly as a guide for persons with a high level of technical competency. I do not provide a warranty of any kind for the quality of specimens prepared using it, and I cannot be held responsible in any way. Do not use this guide if you are unqualified or unsure. Read the MSDS for all chemicals and reagents before use, and use the correct personal protective equipment. Fear osmium, because it can kill you.

Generic Benchtop Specimen Preparation:
Step
Reagent
Time
Primary Fixation
4% PFA in Buffer
1 hour
Secondary Fixation
2.5% GA in Buffer
1 hour
3-5x Buffer Wash
0.1M Buffer
10 min
Tertiary Fixation
1% OsO4 (aq) in Buffer
1 hour
1-2x Buffer Wash
0.1M Buffer
10 min
3x Water Wash
MilliQ Water
10 min
En Bloc Staining
2% Uranyl Acetate
1 hour
3x Water Wash
MilliQ Water
10 min
Dehydration
50% Ethanol
10 min
Dehydration
70% Ethanol
10 min
Dehydration
90% Ethanol
10 min
Dehydration
100% Ethanol
10 min
Dehydration
100% Ethanol
10 min
Dehydration
100% Transition Solvent
15 min
Dehydration
100% Transition Solvent
15 min
Infiltration
Solvent:Resin (3:1) [25%]
1 hour
Infiltration
Solvent:Resin (1:1) [50%]
1 hour
Infiltration
Solvent:Resin (1:3) [75%]
1 hour
Infiltration
Resin 100%
1.5 hour
Embedding
Resin 100% @ 60-70°C
24 hour
Common acronyms:
PFA = Paraformaldehyde
GA = Glutaraldehyde
OsO4 = Osmium Tetroxide

Reagent Note:
The buffers, transition solvents and resins are often tailored to specific projects. A good starting point would be 0.1M Phosphate Buffer, Acetone and Epon-Araldite (aka. Procure-Araldite).