Fig. 2: Homogeneous and deep staining with INSIHGT.

a Experimental steps and principle of INSIHGT for immunostaining. Top row: Tissue is infiltrated with antibodies and a weakly coordinating superchaotrope ([B₁₂H₁₂]^2-, purple dodecahedron) in the 1st staining solution and then transferred into the 2nd solution containing a complexation agent (CD, red ring). Bottom row: The molecular principles of INSIHGT. Weakly coordinating superchaotropes prevents antibody-antigen interactions, removing penetration obstacles. After homogeneous infiltration, subsequent γCD infiltration complexes the [B₁₂H₁₂]^2- ions, allowing deep tissue immunostaining. Reproduced with permission from Illumos Limited. b Benchmark results of four buffers used in deep immunostaining. Enlarged views of smaller areas are shown in insets. Parvalbumin (PVALB) immunostaining signals on cut surface: magenta, bulk-staining signal; green: cut-staining PVALB signal (refer to Supplementary Fig. 1). c Quantification of bulk:cut-staining signal ratio against penetration distance for segmented cells. Each dot represents a cell. Lines are single-term exponential decay regression curves. The signal decay distance constants (τ) are shown in Supplementary Table 1. Hypothetical ideal method performance is shown as a gray line (τ→0+). d Correlation of INSIHGT signal with reference (cut-staining intensity) signal, illustrating 3D quantitative immunostaining. r: Pearson correlation coefficient. e Timeline illustration for a whole mouse brain processing experiment with the different benchmarked methods (drawn to scale). *indicates methods where in principle the use of secondary antibody Fab fragments can lead to halved immunostaining times.