RATDISCO, A NOVEL TISSUE CLEARING AND IMMUNOLABELLING PIPELINE FOR LARGE RAT BRAINS

Understanding brain function requires three-dimensional (3D) mapping of neurochemical architecture. Traditional two-dimensional (2D) methods, including manual sectioning, stereology, and cell quantification, remain widely used but limit our ability to capture the full 3D organisation of brain structures.
Advances in light-sheet microscopy enable whole-organ imaging but require optical tissue clearing. We investigated brain-wide alterations in rat models of autism spectrum disorders (ASD). The large size of rat brains presents challenges for antibody penetration and transparency. To address this, we developed RatDISCO, a solvent-based clearing method that incorporates antigen retrieval to enhance antibody penetration in adult rat tissue.
RatDISCO consists of 4 steps to achieve immunolabelling and optical clearing of adult rat brains. 1. Antigen retrieval to expose antigens that are masked by aldehyde fixation/crosslinking. 2. Methanol de/rehydration and permeabilisation to allow deep antibody penetration. 3. Immunolabelling. 4. Delipidation and refractive index (RI) matching. Ethyl cinnamate (ECi) is used for tissue storage and light-sheet microscopy imaging.
To quantitatively compare antibody penetration, we tested FOXP2 immunolabelling between RatDISCO and another solvent-based method: iDISCO+. Using commercially available software (ArivisVision4D), we created a custom 3D atlas of the rat amygdala to register and quantify FOXP2-immunolabelled nuclei in light-sheet data. RatDISCO significantly improved immunolabelling and antibody penetration in large rat brains compared to iDISCO+, yielding higher FOXP2 cell densities in the amygdala.
Furthermore, we demonstrated RatDISCO utility for labelling neuronal subtypes, glial cells, and vasculature in adult rat brains using immunohistochemistry, virally-labelled circuitry, and tested its compatibility with other species, including mice and human-derived brain organoids.