A Scale CUBIC-based clearing protocol preserves fluorescence in mouse rib bone and cartilage

Abstract Tissue clearing has emerged as a critical method for observing fluorescent proteins in animal models. This technique allows for imaging by making tissues transparent, while preserving the natural structure of biological samples and removing the need for labor intensive tissue sectioning. Conventional tissue clearing methods can be classified into three primary types: hydrophobic, hydrophilic, and hydrogel-based, each possessing unique benefits and drawbacks.1 Hydrophobic techniques, like BABB, 3DISCO, and PEGASOS, create highly clear samples but frequently lead to tissue contraction and may lead to diminished fluorescence. Hydrophilic techniques, such as the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails) series, provide improved preservation of tissue integrity but might fall short of achieving complete transparency. Hydrogel-based methods such as CLARITY and ELAST preserve biomolecules, but the protocols may be intricate and expensive. Clearing skeletal tissues such as bone and cartilage can be especially difficult because of their dense extracellular matrix which may have abundant collagens, high mineral composition, and dense obscuring bone marrow. In addition, clearing procedures frequently do not provide adequate transparency without damaging proteins that fluoresce. To overcome these constraints, we created a clearing protocol based on published Scale CUBIC techniques to be used on adult mouse rib bones.2 Our method does not entail decalcification and improves transparency in about 48 hours. Utilizing transgenic mice, we show that this method effectively maintains both endogenous fluorescence and tissue morphology. This enhanced approach offers a dependable technique for imaging hard tissues such as rib bones in transgenic mouse models.