Whole Mount Preparation of Pupal and Pharate Adult Integument

Allow the white puparia to develop 0-8, 12-28, and 40-96 h after puparium formation, and use these stages for whole-mount preparations of histoblast nests and LECs with the following modification of procedures used for larvae (see Notes 7 and 8).

1. Place 0- to 4-h-old scrubbed and cleaned puparia on a depression slide containing a drop of Ringer's solution to make the appropriate cut. Because the puparial case becomes brittle and begins to apolyse from the underlying pupal cuticle by 4-6 h after pupariation and because the separating pupal integument tends to curl down during fixation and subsequent processing, it is necessary to heat specimens which are 5 h or older after puparium formation, as described in Subheading 3.1., steps 2 and 3, before making the following cuts.

2. Cut the pupariated animals either dorso-ventrally in the mid line (resulting in left and right halves) or laterally (resulting in dorsal and ventral halves).

3. Transfer each of these halves to a Stender dish, half-filled with Ringer's solution. Remove the unwanted debris of internal organs by gently squirting Ringer's solution as described in Subheading 3.1., steps 8 and 9. Trim anterior and posterior ends of these halves to prevent potential folding of cuticle during subsequent processing.

4. Fix, process, stain and counterstain, and dehydrate the filets in labeled and separate dishes as described in Subheading 3.1., steps 10-22. Retain the puparial case during these steps so that it can cradle and protect the fragile integument from damage while handling.

5. Remove the puparial case from stages 8 h after pupariation and beyond.

6. Trim off the head and thoracic regions from the abdominal segments.

7. Mount the stained abdominal segments as described in Subheading 3.1., steps 23-27 (see Figs. 3, 7A,B, 8, and 9).

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