Contrasting and Drying of Cryosections

This step is essential for fine structure preservation as well as for producing enough contrast to visualize subcellular detail in the electron microscope. Many different contrasting methods exist and all can be tried using hydrated cryosections mounted on specimen grids. The final choice depends upon the tastes of the operator.
This is the way we do it.

Method

• Make up a 2% solution of methyl cellulose (25 centipoise, Sigma M6385) by stirring the powder into hot water. Methyl cellulose is slow to dissolve and dissolves better in cold water so leave for a few days at 4°C. When fully dissolved, centrifuge at high speed (eg 60000 rpm in a Beckman 60Ti or 70Ti rotor) for 60 min at 4°C. The supernatant can either be removed from over the pellet, or the tubes can be stored in the refrigerator for 2-3 weeks. The methyl cellulose should be removed without disturbing the pellet at the bottom of the tube.
• Mix the methyl cellulose with a 3% aqueous uranyl acetate solution to give a final concentration of 0.3% uranyl acetate (9 part methyl cellulose to 1 part uranyl acetate)
• Put drops of this solution onto a clean surface (parafilm) on ice and float the grids, sections down, on the drops for 10 min.
• Loop off each grid individually from the methyl cellulose-uranyl acetate solution using a large wire loop. A 3.5mm diameter electric drill bit is useful for making these loops.
• Remove excess liquid from the loop using a filter paper.

Notes

• The final thickness of the methyl cellulose is critical for the production of contrast and fine structure preservation. The thickness is controlled by the amount of excess liquid removed from the loop. Films of optimal thickness have gold to blue interference colors.
• An alternative method is to substitute poly(vinyl)alcohol (PVA) for the methyl cellulose. This will produce a positive contrast.

The sections are now ready to be examined in the transmission electron microscope.

Final Contrasting and Drying: After the sections have been labeled with antibodies and colloidal gold, they are washed in water to remove phosphates. They are then floated on drops of 2% aqueous methyl cellulose containing 0.5% aqueous uranyl acetate. Methyl cellulose is more soluble in cold water so the drops are placed on parafilm cooled on ice. After 10 min. the grids are removed individually from the methyl cellulose in metal loops, excess methyl cellulose is removed and the grids are left to dry. The sections are now ready to be dislodged from the loop and examined in the transmission electron microscope.