X-ray Micro Computed Tomography (MicroCT)
Marta Wawrzyniak, Nathalie Weber, Simon Blanchoud
Abstract
This protocol has been successfully used with colonial tunicates. It has been adapted to our needs based on the following publication: " MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues " by Brian D Metscher.
Steps
Fixation
Clean the slide with the colony of your interest. See Cleaning colonial ascidians V.2.
Anesthetize the animals following this protocol. See Whole colony fixation V.1
Fix the animals in 4% PFA at Room temperature . See Whole colony fixation V.1
Wash twice in 3.3x PBS for 0h 30m 0s .
Rinse tree times with 1x PBS and leave in 1x PBS for 1h 0m 0s .
Staining
Detach the colony with a razor blade and put in a 2mL tube containing 1mL PTA /0.6mL IKI/ 0.4mL Ethanol.
Incubate for 96h 0m 0s at Room temperature on linear shaker (low speed), protect from light.
Rinse quickly 3-4 times with 1x PBS.
Mounting
Prepare 10mL of 0.5% volume low melting agarose.
When the agarose has cooled down a bit, pipette 125-150µL if using a 200ul tip or 750-800µL if using a 1mL tip (the tip size depends on the animal size).
Carefully place your colony vertically inside the tip (it should be as flat as possible, avoid folds).
Pipette some more agarose in order to cover the animal entirely.
Place the tip into a beaker filled with ice-cubes so that the agarose becomes solid.
Cover the tip with fixing paste.
Store at 4°C or proceed to the next step.
Mount the tip on the tomograph support using fixing paste in such way that it is perfectly straight.
Place it inside the tomograph.
Follow the tomograph's manual and proceed to scan.
After scanning, the tip with the sample can be stored at 4°C for several weeks and eventually can be reused.
