Fig. 5: Benchmarking of ThICK staining.
From: Antibody stabilization for thermally accelerated deep immunostaining
a, Orthogonal views of a benchmarking experiment using anti-MAP2 immunostaining with confocal imaging under ×10 magnification. Insets shown are enlarged views of the centermost regions of the 3D image volumes. Scale bars: main panels, 200 μm; insets, 25 μm. b, Voxel-wise MAP2 signal intensity distribution versus distance from the nearest tissue surface. c, Timelines of deep immunostaining methods that had achieved whole adult mouse brain immunostaining, starting from a perfusion-fixed mouse brain. d, Schematic diagram of a stringent benchmarking experiment workflow. A fixed mouse hemibrain was processed through various deep immunostaining protocols with bulk staining (magenta), and then cut and re-stained with the same antibody with a different fluorophore (cut-staining, green) using conventional immunostaining. See Methods for details. Of the methods shown in c, we compared ThICK staining with iDISCO and CUBIC-HistoVIsion (CUBIC-HV). e, 3D imaging results obtained from the benchmarking experiment described in d. The cut surface is shown as an x–y plane (left square images), with the imaging depths of 200 μm shown as an x–z plane (rightmost rectangular pane). The imaging parameters were kept constant to ensure comparability. To ensure fairness in quantifying tissue diffusion distance, all tissues were cleared using the BABB method to obtain approximately the same degree of tissue shrinkage. Insets show the enlarged views of the white boxed areas in the overview images, which corresponds to a similar distance from the nearest bulk-staining tissue surface. Scale bars: main panels, 100 μm; insets, 50 μm. Color bar, pixel intensity. f, The mean signal intensity and distance of segmented cells from e from their respective staining tissue surfaces. Each dot represents a segmented cell and is color coded based on the bulk-staining protocol used. Superimposed lines on the scatter plots are segmental linear regression lines. Histograms of the distributions are shown alongside the scatter plots. ****two-tailed P < 0.0001 (unpaired t-test with Welch’s correction, left; Brown–Forsythe ANOVA test, right).
