Genetically encoded barcodes for correlative volume electron microscopy
brains. HEK293T cells were collected with Accutase 36 h post-transfection and pelleted by centrifugation. As an initial demonstration for multiplexed EMcapsulin detection across different cells, cell suspensions of HEK293T cells transiently expressing a single class were mixed and pelleted by centrifugation. Following the initial preparation, the material was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 20 min .
The sample was retrieved and loaded into an external sputter coater, where an electron-opaque iridium layer was deposited. Back in the FIB-SEM chamber, a 3D platinum pad with a thickness of 2 µm was placed onto the block in proximity to the region of interest. Subsequently, a tracking pattern was milled in the deposited pad to simplify image registration. Finally, the platinum pad and grooves were covered with the carbon layer on top.
. Renderings of the acquired FIB-SEM data were computed either with Imaris or with Dragonfly software .
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