라이브 이미징을 위한 피브린 혈전을 가진 드로소필라 조직의 정화 응용 프로그램

This protocol described sample immobilization using fibrin clots. Samples are transferred to a drop of fibrinogen containing culture medium on the surface of a cover slip, after which polymerization is induced by adding thrombin. Under a dissection microscope, use a brush to transfer L3 larvae into a nine well borosilicate glass dish containing PBS.

Stir to detach most of the fly food from the larvae, and transfer it to another well containing culture medium. Use forceps to grasp a larva across its entire diameter in the middle of its body length, and transfer it to another well of the borosilicate plate containing 200 microliters of fresh culture medium. Do not release the larva.

To cut the larva in half across its entire diameter, either grind the grasped area with lateral movement of the forceps tips, or slide one tip of another pair of forceps between the two forceps tips holding the larva. Use the forceps to hold the larva by its cuticle, and another pair of forceps to peel apart the cuticle without pulling on the brain. Repeat this until the nerves originating from the brain are visible, and the organs connecting the brain to the mouth parts can be accessed.

While holding the nerves originating from the brain with forceps, cut the connection between the brain to the mouth parts with the other pair of forceps, separating the brain from the rest of the cuticle. Hold the brain by the axons coming out of the ventral nerve cord and pluck the surrounding organs by gently pulling them away from the brain. Then grasp the connection between the eye imaginal discs and the brain with the other pair of forceps, and cut this connection without pulling on the brain.

Make sure that the brain is appropriately cleared of other tissues and not damaged. Discard the brain if it shows signs of damage. Pipette the coating solution with a P20 in and out to coat the pipette tip, then aspirate the brain with the coated tip along with three microliters of medium, and pipette it out in another well containing 200 microliters of clean culture medium.

Repeat this process until enough brains are dissected, occasionally replacing the culture medium of the well in which the dissections are performed. Use a P20 pipette with a coated tip to transfer the dissected brains into another well of the borosilicate plate containing 200 microliters of culture medium and fibrinogen. Aspirate one brain along with nine microliters of culture medium and fibrinogen.

With the end of the tip almost touching the cover glass bottom of the cell culture dish, pipette out the contents so that the culture medium touches the cover slip immediately after exiting the pipette tip. Use one tip of a pair of forceps, or a closed pair of forceps, to gently push and position the brain within the culture medium and fibrinogen drop. If the ventral part of the brain has to be imaged, induce fibrinogen clotting to properly orient the brain within the clot.

Push the brain to one side of the drop. Touch the edge of the drop on the opposite side of the brain with the pipette tip and add one microliter of thrombin. Wait for one to two minutes for the fibrinogen to start polymerizing, resulting in the formation of a cloudy precipitate at one side of the drop, then gently push and tuck the brain into the fibrin clot, making sure that the ventral side is as close as possible to the cover slip without deforming the brain.

Pipette out one microliter of thrombin solution close to the side of the brain not tucked into the fibrin clot. Wait for two to three minutes for the second fibrin clot to set, then press on the edges of the clot to make it adhere more strongly to the cover slip, taking care not to deform the brain. If the brain appears to be too far from the cover slip, bring it closer by pressing the fibrin clot closer to the brain.

To induce the fibrin clot for imaging the dorsal part of the brain, position the brain in the center of the drop, dorsal part facing the cover slip. Using a P2 pipette, touch the edge of the drop on the opposite side of the brain with the pipette tip and pipette out one microliter of thrombin. Wait for two to three minutes for the fibrinogen to start polymerizing, resulting in the formation of a cloudy fibrous precipitate, then press on the edges of the clot to make it adhere more strongly to the cover slip, taking care not to deform the brain.

With the end of the tip positioned about 0.5 centimeters above the clot, gently pipette 390 microliters of culture medium without fibrinogen drop wise on top of the clot. Do not add the culture medium on the sides of the clot, as it may detach it from the cover slip. To wash out the excess thrombin, remove 300 microliters from the dish, then add 300 microliters of clean culture medium, making sure that the clots remain fully immersed.

To avoid temperature changes causing changes of focus, keep the solution with the reagents at the same temperature as the culture dish. Remove the lid of the culture dish without displacing the dish itself. For easier removal, place the lid of the 35 millimeter dish upside down on top of the dish before imaging.

Position a P1000 pipette tip close to the surface of the medium inside the culture dish, and gently release the adequate volume of reagent solution onto it, without generating strong fluxes that could detach the clots. To homogenize the solution within the culture dish, use a P200 pipette to slowly pipette a small amount of the solution in and out five times. Replace the lid without displacing the culture dish and resume imaging.

When one NAPP1 was added to fibrin immobilized larval brains expressing GFP-tagged Bazooka, brains carrying an analog sensitive mutation of aPKC, displayed a contraction of the neural epithelium, as well as bright clusters of Baz in neuroblasts and their progeny. Neuroblast kept dividing throughout the time-lapse, indicating that the tissue remained healthy. And brains showed little drift despite the culture medium change.

Similarly, application of one NAPP1 to ovarials expressing GFP-tagged Bazooka induced the contraction of analog sensitive aPKC mutant follicular cells but not of controls. A hyper stack displaying both lateral and focused drift was first corrected for lateral drift, then focus drift, resulting in a substantial reduction of the movements observed in the original hyper stack. When attempting this protocol, it is important to tuck to brain into the partially polymerized fibrin clot while the clot is still malleable but solid enough to stably hold the brain.

Experiment with manipulation of empty clots and gently probe the clot while it is polymerizing to check how sturdy it is.