SHIELD-Processed Organoid Staining

SHIELD-processed and cleared organoids were stained using an adapted version of the eFLASH protocol^{27 (link),44 (link)}. We incubated organoids in eFLASH sample overnight at room temperature. Organoids were then placed in the SmartLabel system (LifeCanvas) with 1.4 mL sample buffer in the sample cup. For SCOUT pipeline, we added 6µL Syto16 (1 mM solution, ThermoFischer #S7578), 15 µg goat anti-SOX2 antibody (R&D Systems #AF2018), 10 µg Fab fragment anti-goat IgG Alexa Fluor 594 (Jackson ImmunoResearch #805-587-008), 30 µg rabbit anti-TBR1 Alexa Fluor 647 (Cell Signaling Technology #45664S). Additional antibodies used for whole organoid staining are anti-β3-tubulin (mouse monoclonal, Biolegend #657408), anti-MAP2 (mouse monoclonal, Biolegend #801803) and vimentin (rabbit monoclonal, Cell Signaling Technologies #45664). All antibodies used in this study are listed in Supplementary Table 1.

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Albanese A., Swaney J.M., Yun D.H., Evans N.B., Antonucci J.M., Velasco S., Sohn C.H., Arlotta P., Gehrke L, & Chung K. (2020). Multiscale 3D phenotyping of human cerebral organoids. Scientific Reports, 10, 21487.

Publication 2020

Syto16 goat anti-SOX2 antibody anti-β3-tubulin anti-MAP2 vimentin

Alexa 594 Alexa fluor 647 Anti antibody Anti igg Antibodies Buffer Fab fragment Goat Map2 Mouse Organoid Rabbit Sox2 Tubulin Vimentin

Corresponding Organization :

Other organizations : IIT@MIT, Broad Institute, Harvard University, Harvard–MIT Division of Health Sciences and Technology, Yonsei University, Institute for Basic Science

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Variable analysis

independent variables

SHIELD-processed and cleared organoids
EFLASH protocol
Incubation of organoids in eFLASH sample overnight at room temperature
Placement of organoids in the SmartLabel system (LifeCanvas) with 1.4 mL sample buffer in the sample cup
Addition of Syto16, goat anti-SOX2 antibody, Fab fragment anti-goat IgG Alexa Fluor 594, and rabbit anti-TBR1 Alexa Fluor 647 for SCOUT pipeline
Additional antibodies used for whole organoid staining: anti-β3-tubulin, anti-MAP2, and vimentin

dependent variables

Staining and visualization of organoids

control variables

Not explicitly mentioned

controls

Positive controls: Not specified
Negative controls: Not specified

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