Block cells with 10% (v/v) normal goat serum (NGS) in 0.1% (v/v) Triton X-100 in PBS and incubate it with primary antibodies in blocking solution .

After a 3-h incubation with the corresponding secondary antibody (Alexa Fluor, Invitrogen), wash the samples and treat with 0.1 mg/ml Acryloyl-X SE solution (Thermo Scientific) in PBS for 3h 0m 0s at Room temperature.

The freshly prepared gelling solution consisted of Stock X solution (8.6% (w/v) sodium acrylate 33% (w/v), 2.5% (w/v) acrylamide 50% (w/v), 0.15% (w/v) N,N´-methylenebisacrylamide 2% (w/v), 11.7% (w/v) NaCl 5 M, and PBS 1X), water, 10% (w/v) TEMED and 10% (w/v) APS stock solution in a 47:1:1:1 ratio.

Perform gel digestion 3h 0m 0s in digestion buffer (0.5% (w/v) Triton X-100, 0.2% (v/v) EDTA 0.5 M, pH 8, 5% (v/v) Tris-Cl 1 M, pH 8, 4.67% (w/v) NaCl and 8 U/ml proteinase K).

Add the gelling solution to each well and covered by a 15-mm coverslip to ensure the formation of a smooth, flat and thin gel.

Incubate coverslips for 1h 0m 0s at 37°C for complete polymerization.

Expand the gel in water for 1h 0m 0s and mount in 10µg/mL poly-L-ornithine-coated coverslips to immobilize the gel for picture acquisition.

Acquire images using a Leica TCS SP8 confocal microscope (Leica, Germany) equipped with a 100× /1.4 numerical aperture oil-immersion objective. For each condition, acquire 5 images from at least three independent experiments.

Analyze Images using Diffraction PSF 3D, DeconvolutionLab2, and EzColocalization plugins in Fiji-ImageJ.

Use GraphPad Prism version 9.0.0 (RRID:SCR_002798) to calculate Spearman’s rank correlation value (ρ) to identify colocalization of fluorescence signals.

The following is a variant of the protocol in case of using midbrain organoid sections:

Fix midbrain organoids and perform immunofluorescence staining as described above.

Treat sections with 0.1mg/mL acryloyl-X SE solution in PBS at Room temperature .

Perform gelation in a 47:1:1:1 ratio of Stock X, 10% (w/v) TEMED, 10% (w/v) APS, and 0.5% (w/v) 4-hydroxy-TEMPO stock solutions.

Perform gel digestion and expansion as described above.

Acquire images using a Leica TCS SP8 confocal microscope (Leica, Germany) equipped with a 100× /1.4 numerical aperture oil-immersion objective. For each condition, acquire 5 images from at least three independent experiments.

Analyze Images using Diffraction PSF 3D, DeconvolutionLab2, and EzColocalization plugins in Fiji-ImageJ.

Use GraphPad Prism version 9.0.0 (RRID:SCR_002798) to calculate Spearman’s rank correlation value (ρ) to identify colocalization of fluorescence signals.