# An Improved SHANEL Procedure for Clearing and Staining Brain Tissue from Multiple Species

## TL;DR

This paper introduces an improved brain tissue clearing method, iSHANEL, that enhances 3D imaging across multiple species by improving transparency and reducing background noise.

## Contribution

The novel iSHANEL procedure improves tissue clearing efficiency and compatibility with nonrodent species.

## Key findings

- iSHANEL significantly enhances tissue transparency and lipid removal in brain tissue.
- The method allows high-resolution imaging of vascular structures with reduced background noise.
- iSHANEL preserves protein immunogenicity and is effective for large tissue samples from pigs and monkeys.

## Abstract

Recent advances in tissue clearing chemistry have revolutionized three-dimensional imaging by enabling whole-organ antibody labeling, even in thick human tissue samples. However, these techniques face limitations, including reduced clearance efficiency in thick tissue blocks, prolonged processing times, and other challenges specific to formalin-fixed human brain tissue, such as autofluorescence due to the presence of lipofuscin, neuromelanin pigments, and residual blood. To address these challenges, we have developed an improved SHANEL procedure, iSHANEL, which is compatible with brain tissue from multiple species, including nonrodents. iSHANEL significantly enhances the tissue transparency. It effectively removes lipids, particularly sphingolipids, from brain tissue across different species. For brain vasculature imaging, it offers the better visualization of vascular structures at high magnification, reducing light scattering and background noise. Moreover, iSHANEL can be effectively applied to large tissue from pigs and cynomolgus monkeys, and it preserves protein immunogenicity well, as evidenced by immunostaining with the microglia-specific marker IBA-1. We also investigated methods to reduce blood vessel autofluorescence. Overall, iSHANEL provides an improved procedure for the high-resolution 3D imaging of brain tissue across multiple species.