Transfer the sample without air bubbles to Eppendorf tubes, and after 30 min at 37°C, centrifuge at 300g for 5 min. After 1 h at 4°C, use a sharp hobby knife to cut off the bottom of the tube containing the cell pellet.

Now coax the cells pelleted in 20% gelatin out of the tube bottom with a needle or awl, and place the pellet in a drop of 2.3 M sucrose on a microscope slide or dental wax cooled with ice. Then cut the sample with razor blades into cubes of maximum 1 mm3, which are cryoprotected overnight (~18 h) at 4°C in 2.3 M PBS-buffered sucrose (prevents formation of ice crystals) with 1% PFA. The cubes with a little cover of sucrose are then mounted in Tissue-Tek on silver sticks and frozen by rapid immersion in liquid nitrogen (Fig. 1). For storage in liquid nitrogen, use cryoplastic tubes with screw caps and small holes made to ensure that the specimen is immersed in liquid nitrogen.

Fig. 1. Preparation of semithin cryosection. Mount the specimen in Tissue-Tek and sucrose on the tip of a silver stick. Remove excess sucrose with filter paper, but leave a thin film of sucrose. Afterward, plug the specimen holder into liquid nitrogen with shaking. Mount the silver stick in the cryochamber cooled with liquid nitrogen, and cut sections of about 500 nm with a glass knife at about -40°C. The sections can be rearranged on the knife using a wood stick mounted with a hair or eyelash. Remove the cryosections from the dry knife with a droplet of 2.3 M sucrose in PBS in a loop. The sections will fly to the approached cooled but not frozen droplet of sucrose. It is important not to let the knife touch the sucrose. The cryosections will spread during the thawing, resulting in thinner sections with fewer wrinkles. The sectioning and this collecting maneuver in particular require practice. By a slight touch, the sections and a part of the sucrose are placed on the microscope slide in the marked circles. The slide is now ready for immunoincubation. Throughout the procedure it is important that the sections never dry.

9. To make semithin cryosections (Fig. 1), prepare a glass knife according to the manufacturer's instructions. Alternatively, a diamond knife might be used. Mount the silver stick in the cryochamber cooled with liquid nitrogen in the cryo-ultramicro-tome, cut sections of about 500 nm with the knife at about -40°C, and transfer the sections from the knife by means of a sucrose droplet in a wire loop to the microscope slides prepared as described in Subheading 3.2. 10. If convenient, the slides can be stored at 4°C in PBS-buffered 1% PFA overnight before immunolabeling.

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