Fig. 2.

Pupal sample preparation for cryosectioning. (a) Placed timed pupae on double-sided tape. Ensure the pupa is secure and dorsal facing upward. (b) 16-h pupa with the first step of the casing removed. (c) Continuing the removal of the case; notice the extra casing removed on either side. (d) The 16-h pupa that is finally prepared is shown where the removal is only necessary around the thorax (e) Samples are aligned onto the spatula, notice that some pupae may roll. Ensure that the pupa has not rolled with the ventral side up (f) A liberal amount of tissue freezing medium immerses the pupa in order to properly stabilize the tissue. Add a sufficient amount of tissue freezing medium to cover the samples, but not too large as a large piece may break when freezing in liquid nitrogen. (g) Prepared sample is placed in deepest corner of Styrofoam box filled with liquid nitrogen. (h) Samples are completely submerged in liquid nitrogen and removed when bubbling is minimal. (i) Samples can now be stored in a 15 mL centrifuge tube in the −80 °C freezer