Thunder imaging system ctr5500

The quantification of vessel-associated oligodendrocytes was performed in mouse brain slices at E20 after double immunostaining with Olig2 (oligodendrocytic lineage) and CD31 (endothelial cells) antibodies (Supplementary Table 1). Z stacks acquisitions were done at 40× magnification using a Leica Thunder Imaging System CTR5500 in three groups: non-electroporated placenta (Ctrl), CRISPR-negative/GFP-positive electroporated placenta (Ctrl_ep), and CD146-CRISPR/Cas9/GFP transfected placenta (CD146-CRISPR). Afterward, Z-stack series of images were loaded into IMARIS imaging software 9.0.2 (Bitplane, Zurich, Switzerland) for 3D reconstruction (Supplementary Videos 1, 2). To validate a vessel-oligodendrocytes interaction, the maximal distance between the center part of Olig2-positive cells and the center part of the vessel was fixed at 10 μm (Brosolo et al., 2022 (link)).

Measurement of the oligodendrocyte density in cortical layers and CC was performed after immunostaining of E20 brain slices using the Olig2 antibody (Supplementary Table 1). Images were acquired at 10× magnification using a Leica Thunder Imaging System CTR5500, and ROIs were defined within the whole thickness of the sensorimotor cortex or by distinguishing the three regions SL, DL, and CC. Fluorometric analysis using the multi-point counting tool of the Fiji software gave access to the number of immunoreactive cells present in the ROI. Density was then determined by a ratio between the number of cells and the ROI area. The analysis was repeated in both hemispheres and in three slices per animal (Brosolo et al., 2022 (link)).