Principles & Protocols

Protocols - TRIAFOL METHOD

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Solutions Required:-

• Chloroform or Acetone -to wash slides O/N or <=3 days
• 0.5% Plastic (Cellulose Acetate/Triafol) dissolved in Ethyl Acetate -to make holey plastic sheet
• Softex 0.1% stock -to make slides hydrophobic
• (Pelex 0.5-1% -to float off plastic, although no longer necessary as we use hydrofluoric acid vapors)

Instructions:-

NOTE: Use forceps/hemostat only to touch slides (no fingers)!

1. Soak slides in CCl4 (chloroform) or acetone for a few hours to 3 days in the glass "slide-container-boxes" (called "staining dishes"), in the fume hood.
2. Pull out the inner glass rack from the staining dish (which holds the slides in the CCLl4/acetone), and let slides dry.
3. Soak slides in Softex-- start with 0.03% solution for 30 mins-- soak 5 at a time in the vertical glass Coplin containers.
4. Rinse slides by transferring to Coplin jar full of water.
5. Pull out of water and observe that the water all flows off, leaving only a few large drops at most-- blot these off with filter paper. If most of the water doesn't flow off-- soak in Softex solution longer to make slide more hydrophobic. Let slides dry. Slides alright to use for a few days, but immediately is better.
6. Put 1 slide onto a metal plate that has been pre-cooled and is in the fridge-- you need to empirically test the length of time from ~10-30 seconds that you leave the slide on the metal plate in the fridge.
7. Pull out glass slide and immediately (or vary the time here too) squirt a nice continuous smooth layer of plastic solution ~1.5 ml of 0.1-0.3% plastic onto EACH side of the glass slide-- try to use a wide-tipped glass pipette to apply the plastic in one nice wide stream by squirting across the top edge of each side in one motion to let the plastic flow over the slide in one big even sweeping motion.
8. Do this over a glass petri dish lined with tissue to collect the run-off and dab the end of the slide to blot the accumulated solution at the bottom edge.
9. Let the slide dry-- you should see it turn from cloudy to more transparent as the water droplets evaporate.
10. Go to an optical microscope and look/note down size and distribution of holes (can put an EM grid on the slide to compare hole size/distribution relative to a grid)-- mark good areas on the edge of the plastic to indicate where to put grids when floating. If plastic isn't perfect, hypothesize what variables to adjust to achieve desired characteristics:

VARIABLES TO TEST:-

a Concentration of Softex a Concentration of Plastic a Time in fridge a Time out of fridge (even to the point of letting some drops evaporate) a Different hydrophobic solutions a Different humidity level

• Follow other instructions on how to finish off making the holey grids-- i.e. floating plastic and carbon-coating, dissolving plastic etc.

II) FORMVAR/GLYCEROL METHOD

1. Make a 0.3-0.5% solution of Formvar in Chloroform (0.6-1% for 200cc).
2. Prepare a mixture of glycerol and water (50-50)-- agitate enough to mix well.
3. Mix 50cc of the Formvar solution with 20 drops of water-glycerol mixture. Agitate vigorously by hand until milky, then with a sonicator for 2-5 mins on high setting until the container gets warm. NOTE:- More water-glycerol (100-120 drops) gives holes of greater size.
4. Dip a clean microscope slide into the solution. Dry for a few seconds and look at the film in the optical microscope.
5. If satisfied, float the film on water and proceed as usual. Pick up film and grids with filter paper. Dry. Etch film with methanol for 10mins. Dry.
6. Proceed as usual with carbon evaporation.
7. Wash film away with ethylene dichloride 1 hour. Dry.

• Set ALLOWTOPICCHANGE = CemfacGroup