Cryostat Sections

Cryostat sections are the method of choice if the photoreceptor cells of the compound eyes, which strongly express the clock genes, are the focus of interest. The following sections describe the preparation of the flies for cryostat sections as well as the principal sectioning technique. Note that many cryostats are designed for cutting larger tissue from vertebrates. Therefore, the cryostat chuck is often too large for small insects. For Drosophila heads a cryostat chuck with a diameter of 5 to 7 mm is optimal, and it might be necessary to ask a workshop to make such a chuck.

3.2.1. Preparation and Fixation of Flies for Cryostat Sectioning

1. Remove head, proboscis, and air sacs of flies as described for the whole-mount preparation (see Note 11).

2. Fix the dissected heads in PFA for 3 h.

3. Wash three times for 10 min in PBS at room temperature.

4. Incubate the heads in 25% sucrose (in PBS) overnight at 4°C.

5. Put a drop of Tissue Tec on a slide and submerge one head in it; it is important that the whole head is bathed and that no air bubbles stick to the head.

6. Put a large drop of Tissue Tec on the cryostat chuck (no air bubbles). Using a long pair of forceps submerge the base of the cryostat chuck into liquid nitrogen. Only the base of the drop should freeze (white color); the top must stay fluid (Fig. 2A).

7. Remove the cryostat chuck from the nitrogen and place one head into the fluid Tissue Tec; orient it in the way shown in Fig. 2A.

8. Slowly submerge the cryostat chuck into nitrogen until the Tissue Tec drop is completely frozen.

Fig. 2. Cryostat sectioning of Drosophila heads (horizontal sections). (A) Heads are embedded and oriented in Tissue Tec. (B) Trimming of the frozen block. (C) Sectioning and transferring of the sections to a subbed slide.

9. Put the cryostat chuck into the cryostat (-20°C), but wait some minutes before cutting as the block with the sample might be still too cold. Also put some subbed slides into the cryostat.

3.2.2. Sectioning With Cryostat

1. Cut the block with the microtome until the surface becomes flat. Trim the block with a razor blade to an oblong shape (Fig. 2B). It is important to make sure that the bottom edge and the vertical side of the block are, respectively, parallel and perpendicular to the knife edge.

2. Adjust the antiroll plate on the knife and cut 6- to 10-^m sections; they should form a ribbon.

3. Removed the antiroll plate and pick up the ribbon with a cold slide (Fig. 2C).

4. Thaw the ribbon by touching the slide from below with a finger, and then keep the slide at -20°C for an additional 15 min.

5. Remove the slide from the cryostat and allow it to air-dry for 30 min (see Note 12).

6. Circle the sections with a wax pencil and arrange the slide(s) horizontally on two parallel glass bars in a humid chamber (see Note 13).

7. Proceed with the immunostaining.

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