Preparation of Fixed Drosophila Oocytes for Immunostaining: A High-Throughput Method to Fix and Remove the Outer Membrane

- Begin by pulsing anesthetized Drosophila along with buffer in a blender to break up flies into small pieces. Filter the mixture through a mesh to remove the large body parts. Then, allow the eggs to sink while the larger fragments remain on the surface and can be removed.

Repeat this process using a smaller mesh to remove additional debris. Collect the eggs in a vial, and if the eggs are to be treated with a drug, do so at this point. Next, remove the liquid and replace it with fixative. Add heptane to help the fixative penetrate the membrane surrounding the egg.

Then, wash away the fixative with PBS. To stain the oocytes, remove the protective outer membranes to allow for antibody penetration. Pipette the oocytes onto the frosted part of a glass slide. Place a coverslip over the eggs and gently roll the coverslip in a back-and-forth motion to mechanically separate the chorion and vitelline membrane from the egg. The oocytes are now ready to be stained.

In the example protocol, we will be preparing oocytes in meiosis I to stain and visualize the spindle apparatus.

- To begin, precut the inside of a 5-milliliter tube and Pasteur pipette with PTB to prevent oocytes sticking to the