Anti map2

15 mm coverslips with neurons were fixed in 4% PFA, permeabilized with 0.1% Triton X-100 and incubated overnight with primary antibodies diluted in 3% BSA/PBS at 4°C: (anti-MAP2 [1:2,000, 822501; BioLegend], anti-PSD95 [1:1,000, 75-028; Neuromab]). Next day coverslips were incubated for 1 h at RT with secondary antibodies (Alexa-488 α-chicken [1:1,000, A-11039; Thermo Fisher Scientific], Alexa-555 α-mouse [1:1,000, A-31570; Thermo Fisher Scientific]) and DAPI (1:5,000). Finally, coverslips were mounted with ProLong (Thermo Fisher Scientific) and imaged at confocal microscope Nikon A1R+ with 60×/1.4 Plan Apochromat. Fluorescence quantification was done with FIJI software. A 12% threshold was applied over the MAP2 signal to create a mask. This was used over PSD95 channel to quantify a specific signal within neurons.

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.

Cells were washed with PBS 1× at room temperature (RT) and fixed in 4% paraformaldheide (PFA) diluted in PBS 1× for 10 min at RT and in the dark. Then, PFA was replaced by PBS 1×, and samples were kept at 4 °C until processing for immunofluorescence analysis. On the day of the experiment, cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS 1×, then blocked for 15 min with 3% Bovine Serum Albumin (BSA) in PBS 1×. Coverslips were incubated with the primary antibodies diluted in PBS 1× for 2 h and at RT: anti-GFAP 1:500 (MAB3402, Merck MilliporeSigma); anti-MAP2 1:500 (822501, Biolegend, San Diego, CA, USA); anti-Oligo2 1:500 (R&D Systems, Minneapolis, MN, USA). Next, coverslips were washed 3 times with PBS 1× and incubated for 1h with an Alexa555-conjugated anti-mouse antibody (1:1000; Thermo Fisher Scientific) and Alexa488-conjugated anti-chicken antibody (1:1000; Thermo Fisher Scientific). After a 5 min DAPI staining (1 µg/mL in PBS 1×, Merck MilliporeSigma) and 3 washes with PBS 1×, coverslips were finally fixed using Mowiol-DABCO gel mounting agent (Merck MilliporeSigma).
The preparations were analyzed with an LSM 710 confocal microscope system (Zeiss, Oberkochen, Germany). Zeiss imaging software was used to analyze the confocal images.