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Large-scale connectomics requires dense staining of neuronal tissue blocks for electron microscopy (EM). Here we report a large-volume dense  en-bloc  EM staining protocol that overcomes the staining gradients, which so far substantially limited the reconstructable volumes in three-dimensional (3D) EM. Our protocol provides densely reconstructable tissue blocks from mouse neocortex sized at least 1 mm in diameter. By relaxing the constraints on precise topographic sample targeting, it makes the correlated functional and structural analysis of neuronal circuits realistic.

Large-volume en-bloc staining for electron microscopy-based connectomics