Fig. 4: Application of SPEARs in ThICK staining of human brain and mouse whole-brain tissues with different tissue clearing techniques.

a, Protocol for a 5-mm-thick human brainstem block ThICK-stained with tyrosine hydroxylase (TH) SPEAR^py. b, Image stack overview with segmented neurons color coded by cell volume. Scale bars, 500 μm. c, Magnified x–z view of the white boxed area in b. Scale bar, 100 μm. d, TH-positive neurons in optimized conventional immunostaining with 100 μl anti-TH antibody for 2 weeks (left) versus ThICK staining with 30 μl TH SPEAR^py for 2 days (right). Scale bars, 500 μm. e, Distance of segmented neurons in d from the nearest tissue surface in conventional versus ThICK staining. Upper panel: each data point represents one segmented neuron (n = 707 and 2,828 for anti-TH antibody and TH SPEAR^py, respectively). The black lines represent the median and the error bars represent the interquartile range. Lower panel: cumulative distribution of the distance. f, Protocol for a SHIELD-protected whole adult mouse brain ThICK-stained with ChAT SPEAR. g, Overview of the rendered image volume. Excerpted cross-sectional and volume rendering views are shown in h–k, with insets showing magnified views. Scale bar, 2 mm. h, Volume rendering of cholinergic fibers traversing the basolateral amygdala (Am) with the internal capsule (IC) and external capsule (EC) included. Scale bar, 200 μm. i, Cholinergic neurons (arrowheads) in hippocampal CA1. Scale bar, 100 μm. Inset: magnified view of the white boxed area. Scale bar, 20 μm. j, Pedunculopontine nucleus (highlighted with dotted line). Scale bar, 200 μm; inset, 20 μm. k, Horizontal diagonal band of Broca (HDB). Upper panel, 100 μm z-MIP; lower panel, excerpted single section. Scale bars: 200 μm (upper), 100 μm (lower). acom, anterior commissure. l, Whole-brain tractography of cholinergic fibers, color coded for passage through major anatomical structures (upper panel), origins from major cholinergic cell groups or passage through major tracts (lower panel). m, Protocol for whole adult mouse brain ThICK-stained with parvalbumin SPEAR^py with iDISCO tissue processing and BABB clearing. n, Overview of the rendered image volume. Scale bar, 1 mm. o–r, Excerpted views, including reticular thalamic nucleus (RT, o), cortex and hippocampus (Ctx and Hp, p), periaqueductal gray region (PAG, q), and cerebellum (inset, r). Scale bars: o–q, 200 μm; r, 1,000 μm. In r, the spatial locations of all automatically identified PV-positive interneurons in a cerebellar hemisphere volume are shown, with each neuron represented by a spot. Inset, 3D-rendered image.