Preparation of Fixed Drosophila Oocytes for Immunostaining: A High-Throughput Method to Fix and Remove the Outer Membrane
Transcrição
– Begin by pulsing anesthetized Drosophila along with buffer de a blender para break up flies into small pieces. Filter the mixture through a mesh para remove the large body parts. Then, allow the eggs para sink while the larger fragments remain on the surface and can be removed.
Repeat this process using a smaller mesh para remove additional debris. Collect the eggs de a vial, and if the eggs are para be treated with a drug, do so at this point. Next, remove the liquid and replace it with fixative. Add heptane para 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 para 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 de a back-and-forth motion para mechanically separate the chorion and vitelline membrane from the egg. The oocytes are now ready para be stained.
In the example protocol, we will be preparing oocytes de meiosis I para stain and visualize the spindle apparatus.
– To begin, precut the inside of a 5-milliliter tube and Pasteur pipette with PTB para prevent oocytes sticking para the surfaces. Add 100 milliliters of Rob's buffer para a blender and then add 100 para 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 de the coated pipette para 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 de the coated pipette. Let the rinse settle de the 5-milliliter tube for three para 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 para 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 para 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 para 1,000 oocytes para 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.
