Chorion and Vitelline Membrane Mechanical Removal: A Method to Prepare Drosophila Oocytes for Direct Observation
DEŞİFRE METNİ
– Begin with mature oocytes from fixed ovaries in a saline solution. Mature oocytes bear eggshells consisting of an outer chorion and inner vitelline membrane.
To mechanically remove the chorion and vitelline membrane, place the oocytes between the frosted parts of two pre-treated slides. Move the top slide videodan straight back-and-forth motions için create friction that rolls the oocytes.
Now, move the top slide at a slight angle için roll the oocytes videodan another direction. Avoiding circular motion, increase this angle videodan short increments until the top slide moves perpendicular için the bottom slide. This movement mechanically removes the chorions and vitelline membranes, enabling access for probes or stains into the oocyte.
Oocytes without chorions will appear long and thin, and those without vitelline membranes will have pointed ends. To separate the cleared oocytes from the surrounding debris, transfer the sample mix için a tube, and allow the oocytes için settle için the bottom while the debris floats at the top and can be removed.
In this protocol, we will remove chorion and vitelline membranes from mature Drosophila oocytes.
– To separate the late-stage oocytes, first, add 1 milliliter of PBSBTx için a shallow dissecting dish. Then, use a P200 with a BSA-coated tip için transfer fixed ovaries into the shallow dish. Pipette the ovaries up and down with the BSA-coated pipette tip için dislodge the mature oocytes from the less mature oocytes.
When late-stage oocytes are sufficiently separated, transfer all the tissue için a 500-microliter microfuge tube. Remove excess liquid with a pulled Pasteur pipette, leaving about 150 için 200 microliters videodan the tube.
To prepare for oocyte rolling, pre-wet a deep-well dish with 200 microliters of PBSBTx. Cover the dish and set it aside.
Obtain three frosted glass slides and set slide three aside. Next, gently rub the frosted glass regions of slides one and two together. Rinse them videodan deionized water için remove any glass shards and dry with a disposable wipe. Coat the frosted regions of slides one and two with PBSBTx by adding 50 microliters of PBSBTx için one slide and rubbing this region with the other slide. Remove the liquid with a disposable wipe and place the slides under a dissecting microscope. Keep the frosted regions of slides one and two videodan contact with slide three supporting slide two.
To roll the oocytes, first, pre-wet a P200 pipette tip videodan PBSBTx and disperse the oocytes videodan the microfuge tube by pipetting up and down. Transfer 50 microliters of liquid containing the oocytes için the center of the frosted glass part of slide one. Lift slide two için do this.
Slowly lower slide two until the surface tension of the liquid creates a seal between the two frosted glass regions. There should be enough liquid için cover the frosted area, but none should be seeping out. Then, hold the bottom slide one videodan place with one hand and use the other hand için move the top slide two back and forth videodan a horizontal direction, keeping slide two level and supported on slide three.
Perform under a microscope for easy visualization of oocyte movements and progress. After a few movements videodan the horizontal direction, slightly change the angle of movement. In multiple increments, gradually increase this angle için 90 degrees until movement of the top slide two is perpendicular için the starting direction. Note that empty chorions will be visible videodan the liquid, and oocytes lacking chorions will appear longer and thinner.
– When rolling the oocytes, ensure that the direction of rolling is always videodan a straight line and never a circular motion.
– Repeat the rolling about 7 için 10 times until the solution becomes slightly cloudy. Stop rolling when the majority of oocytes appear için have lost their vitelline membranes.
Gently lift the top slide two, dragging one of its corners için the center of the frosted region at the bottom slide one so that the rolled oocytes accumulate videodan the center of the frosted region. Rinse the oocytes from both slides with PBSBTx into the deep-well dish containing PBSBTx.
Clean slides one and two with ultrapure water. Dry with a disposable wipe and reset. Repeat these steps until all the oocytes of the same genotype have been rolled. This usually requires three için four rounds of rolling per genotype.
To remove debris after rolling, add 1 milliliter PBSBTx için a 15-milliliter conical tube. Swirl the liquid için coat the sides of the tube. Using a PBSBTx-coated P1000 pipette tip, transfer the rolled oocytes from the deep-well dish için the conical tube containing 1 milliliter of PBSBTx. Add an additional 2 milliliters of PBSBTx için the conical tube containing the oocytes.
Hold the conical tube against a dark background için see the opaque oocytes as they sink. After letting the oocytes settle için the bottom, use a P1000 için remove the top 2 milliliters of solution containing debris and discard.
After repeating the step for a total of three rounds of debris removal, use a PBSBTx-coated P1000 pipette tip için transfer the oocytes back için the original 500-microliter microfuge tube. 20 için 25 females should yield approximately 50 microliters of rolled mature oocytes.
