Fig. 1: High-contrast staining protocol for brain samples 2 mm to 1 centimeter in size, applied to human cortical tissue and adult mouse whole brains.
a, Overview of en bloc staining protocol development: reduced-osmium-TCH protocols1,2,3,25 for samples up to about 200–250 μm in thickness and a more recent6 1-mm protocol, compared to the protocol reported here (gray boxes) enabling gradient-free en bloc staining of samples 2 mm to 1 centimeter in size (sufficient to cover the depth of the human cortex (Fig. 2c–e) and entire adult mouse brains). b, Staining of a sample 2 mm in size using the 1-mm protocol6 (top) and our protocol (bottom) (SEM images at a voxel size of 11.24 nm; dose, 47 e− nm−2). c, High-contrast staining of whole mouse brains. High-resolution EM images acquired from the superficial cortex to subcortical tissue; approximate positions are indicated in the μCT overview (left). High-resolution SEM images (at a voxel size of 5.62 × 5.62 nm2; 113 e− nm−2). μCT data of whole brains: https://wklink.org/7742 and https://wklink.org/7789. d, Illustrations and examples of key challenges of hemisphere and mouse whole-brain staining. Yellow arrows point to sites of intracellular overextraction with loss of cytosolic components (left) and breakages of the sample at microscopic (middle) or macroscopic (right) scale. e, ATUM cutting test at the center of a fully stained and fully embedded mouse whole-brain sample (sample W3, Spurr’s resin, Supplementary Table 1). Middle, sketch and picture of ATUM cutting at sample center, trimmed to about 4 × 2 mm2 for ATUM cutting. Right, sketch of the location of two artifact-free ATUM test series at the exposed surface and after an additional depth of 1 mm (Supplementary Video 1).
