Fig. 6: LGTV replication visualized in ex vivo mouse brain using cryo-ET.

A Workflow for cryo-ET of ex vivo mouse brain. LGTV-infected brains from Ifnar^−/− mice were processed differently based on the imaging method. 1. Fixed and cleared brains were immunostained for the viral protein NS5 (orange/red) and imaged using optical projection tomography. Zoomed insets show infected regions of the choroid plexus and its anatomy. 2. For cryo-ET, the choroid plexus from unfixed and unstained LGTV-infected brains was (i) high-pressure frozen, (ii) trimmed using a cryo-ultramicrotome, (iii) FIB milled, and (iv) transferred to a cryo-TEM for cryo-ET. B Slice through a tomogram of LGTV-infected choroid plexus showing viral replication organelles and mature virions in proximity, enclosed within membrane vesicles. C, D Close-up of the areas indicated in (B) along with their corresponding segmentations. E Slice through a tomogram of LGTV-infected choroid plexus showing a bona fide RO with a thicker membrane than the ER, consistent with observations from Fig. 2. F Close-up of the area indicated in (E) along with its corresponding segmentation. B–F Colors as in Fig. 3. Scale bars, 100 nm.