Section Retrieval

Sections are removed from the surface of the knife using a drop of 2.3M sucrose in PBS. The drop, on a small wire loop is placed into the cryochamber and either used immediately to pick up the sections or left to cool down before picking up the sections. If used immediately, the sections will spread out on the drop. This is useful in removing the compression artifact produced during the sectioning procedure (Griffiths et al, 1984).

Too much spreading, however, can damage the morphology of lightly fixed material. The surface tension on the sucrose droplet may pull apart tissue sections or cell pellets. In this case it is advisable to pre-cool the sucrose prior to section retrieval. The amount of section spreading can be controlled by how much the sucrose is cooled.

For picking up semi-thin sections, where the cryo-chamber is at - 60°C, the sucrose droplet must be pre-cooled or the sucrose will stick to the knife surface.

For holding the sucrose droplet, two kinds of loop can be used. Using a small loop will ensure that the sections picked up will always be in the center of the grid. Using a large loop will mean that more sucrose is present in the droplet and will thus freeze slower. However, the position of the sections on the drop may not be clear and sections may be placed inaccurately on the grids.

Once the sections are on the sucrose droplet, remove the droplet from the cryo-chamber, thaw it and place the drop, section side down, onto a formvar/carbon coated specimen grid. If the grid is loosened from the surrounding film then it will stick to the sucrose droplet. This makes it convenient to transfer the grid onto a dish of PBS (or PBS-FCS, or 2% gelatin, or 0.3% agarose with 2% gelatin). Transfer is performed by gently pushing the loop into the liquid so that the grid is perpendicular to the liquid surface. Surface tension will remove the grid from the loop. The grid should now be floating on the liquid surface with the section side down.

It is important that from now on the section side of each grid stays wet and the top side stays dry.

Manipulating the sections: The sections are moved around the knife surface with an eyelash probe to flatten them and arrange them in single layers. They are collected, away from the knife edge, in groups on the knife surface.
Recovering the sections: Groups of sections can be removed from the knife surface using a drop of liquid in a metal loop. The sections attach to the surface of the drop and can be easily removed from the cryochamber. When thawed, the sections are placed onto metal specimen grids which have been coated with formvar and carbon. A routine solution for retrieving sections is 2.3M sucrose. More recently, solutions of methyl cellulose and methyl cellulose with sucrose have been used.
Next step: Immunolabeling.