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 surfaces. Add 100 milliliters of Rob's buffer to a blender and then add 100 to 300 anesthetized flies and pulse three times for one second.

Filter the resultant slurry into a 250-milliliter beaker through a large mesh with pores of around 1500 micrometers. Let the filtrate settle for around two minutes and then aspirate the top layer, removing as many large body parts as possible. Filter the slurry again into a 250-milliliter beaker through a smaller mesh with the pore size of 300 micrometers.

Rinse the first beaker using additional buffer in the coated pipette to collect the remaining oocytes. Let the slurry settle for three minutes and then aspirate all but the bottom 10 milliliters. Pour as much of the 10 milliliters as possible into the coated 5-milliliter tube. Let the slurry settle for around two minutes and then carefully remove the liquid.

Add the remainder of the 10 milliliters of slurry and let the mix settle again before removing the liquid. Rinse any remaining oocytes out of the beaker using additional buffer in the coated pipette. Let the rinse settle in the 5-milliliter tube for three to five minutes.

To fix the ovaries, first, aspirate off all liquid from the tissues, and immediately add 1 milliliter of fix mix. Place the tissues on a nutator for two and a half minutes. Next, add 1 milliliter of heptane, vortex the tube for one minute, and then allow the oocytes to settle for a further minute.

Remove all liquid from the settled tissue, then add 1 milliliter of 1x PBS. Vortex the tube for 30 seconds, and then allow the oocytes to settle for one minute. After that, remove all liquid from the tube and fill it with five milliliters of 1x PBS.

Using the coated pipette, add roughly 500 to 1,000 oocytes to a frosted glass slide. Remove any extraneous body parts or material using forceps. Place a coverslip on top of the tissue, and gently roll the oocytes until all membranes are removed. Dragging the edge of the coverslip across the oocytes works well. Check the progress of membrane removal under a microscope periodically. Too much pressure will destroy the oocytes.