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Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding \u201cSylite\u201d\u2014a probe with outstanding signal\u2010to\u2010background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super\u2010resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. 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