Figure 1. Novel large-scale en-bloc EM staining protocol for dense connectomic circuit reconstruction: protocol comparison.

(a) Screening strategy for brain biopsy samples >1 mm in smallest dimension. Samples were screened after cutting the sample in about half, such that the core of the sample was exposed and could be tested for staining quality. Samples were screened in overview (1), periphery (2), intermediate (3) and core regions (4). (b–e) Scanning electron microscopy images from mouse cortex biopsy samples screened as indicated in a (regions 1–4, left to right columns) and stained with en-bloc protocols from ref. 18 (b, rOTO), ref. 19 (c, rOTO) and ref. 2 (d, PATCO) and this protocol (e) Note the good staining quality in the periphery (first column) in b,c but the strong over-staining in the intermediate region yielding stain uptake of a subset of neurons (b,c) and a substantial staining gradient, which is relieved in this protocol. (e) PATCO provides good stain penetration but stains myelinated neurites only, (d) Scale bars, 200 μm in b–e left column and 2 μm in other columns.