Extended Data Fig. 3: coCATS labeling pattern in relation to synaptic vesicle associated proteins.

STED imaging in the CA3 stratum lucidum after in vivo microinjection into adult mouse brain and transcardial fixative perfusion. CoCATs labeling (gray) was performed with STAR RED-NHS and immunolabeling (orange) of SYNAPTOPHYSIN1, SYNAPSIN1/2, VAMP2 (vesicle-associated membrane protein 2), VGLUT1 (vesicular glutamate transporter, excitatory synapses) and VGAT (vesicular GABA transporter, inhibitory synapses) was performed with AF594. High-intensity coCATS features at putative synaptic cleft regions (pSCRs) are present both at excitatory and inhibitory synapses. Overview images for xy-STED with zoomed views (raw data) and high magnification views both in lateral and axial directions at near isotropic resolution. STED imaging at near isotropic resolution was performed with 80% of STED power with π-tophat phase modulation (z-STED pattern) and 20% of power 4π-helically phase modulated²⁴ or with z-STED alone. N2V was applied to both channels independently. In some cases, 3D-histogram matching (Fiji/ImageJ Bleach Correction) was applied in the immunolabeling channel to compensate for limited antibody penetration. For detailed parameters see Supplementary Table 1. Tests for pSCR location relative to synaptic markers in Extended Data Figs. 3 and 4 were performed for a total of 10 markers across 17 brain slices from n = 6 animals.