Author: Microscopy Laboratory (This procedure can also be used for virtually any material that must be pelleted prior to fixation and thin sectioning) ### Primary fix: - 2% glutaraldehyde (from 25% or 10% stock) in microtubule assembly buffer. - For better contrast of the tubulin, you can add 0.1-.5% tannic acid. ### Secondary Fix: 0.5-1% OsO4 in the same buffer. OsO4 is kept in the refrigerator in a jar and is made as a 4% solution in water. It is very volatile and must be used in the hood. Osmium crystals take about a day to dissolve, so be certain that there is sufficient stock before you start your experiment. 1. Pellet microtubules at 15-20,000 rpm in eppendorf tubes. Try to obtain a small and tightly packed pellet. - Remove supernatant and overlay pellet with glutaraldehyde solution. Fix at room temperature for ~ 1 hour. If necessary, this can be left overnight at room temperature. - If the pellet is large, loosen it from the bottom of the tube by making a small wedge with a Q-tip stick and using the stick to slide the pellet off the bottom. Then cut the pellet into small pieces with a razor blade. - Rinse the pellet with 2-4 changes of fixation buffer without the glutaraldehyde. - Postfix with 0.5-1% OsO4 in the microtubule assembly buffer or in 50 mM HEPES, pH 7.0 for ~1 hr at room temp or on ice. - Rinse the pellet with several changes of water and soak in 1% uranyl acetate for 30 min - overnight, room temperature. NOTE: the pH of uranyl acetate is ~4.0 so your water rinse should remove any PIPES, that would precipitate at low pH, or phosphate buffer, which precipitates as uranyl phosphate crystals. - Transfer the pellet to a glass scintillation vial and rinse with water. Then dehydrate specimen by 10-15 min incubations in acetone:water mixtures of 25%, 50%, 75%, and at least two 100% acetone rinses. - Infiltrate the samples with plastic. Incubate 4 hrs-overnight in 1/3 resin and 2/3 acetone. Remove the resin and add 2/3 resin:1/3 acetone and incubate for another 4-6 hours in the hood on the rotator. Finally, transfer to 100% spurr plastic and incubate for 2-6 hours. - I like to use a reasonable volume of the 1/3 plastic:2/3 acetone mixture, put it on the rotator in the hood with the cap off overnight, and allow the acetone to gradually evaporate in the hood. Be certain that there is enough plastic to cover the specimen and that the acetone does not evaporate and leave the specimen exposed to the air. Then simply remove remaining resin and add 100% resin to the sample and keep on the rotator for another 4-6 hours. - Pour resin in the flat embedding molds, position your sample near the front (pointy) part of each mold, and insert a note at the back of the block to identify it. I usually use a small piece of 3x5 card with notes written in pencil. - Put in 80 degrees oven and bake overnight or for ~48 hrs. Remove and section.