Improved β-catenin detection in spinal cord tissue sections: autofluorescence quenching

Abstract

Experimental studies on spinal cord regeneration are focusing on the windows of opportunity to improve spinal cord microenvironment via spinal-centric repair pathways. One pathway of particular interest is the Wnt/β-catenin signalling pathway which plays a vital role in axonal guidance, synaptic assembly and function, neuronal survival and connectivity after spinal cord trauma to induce repair. Upregulation of β-catenin expression is often taken as evidence of regeneration mechanisms through the Wnt/ β-catenin pathway. However, these studies may not have optimised the staining protocol for β-catenin to enable accurate detection of the protein. Given possible issues with the background or endogenous tissue autofluorescence, there is a need to optimise the protocol further to allow better visualisation of β-catenin. So far, there are no studies which report optimising spinal cord tissues for β-catenin to reduce autofluorescence, and as β-catenin is widely used in spinal cord injury (SCI) and other spinal cord tissue studies, thus it is an important issue to address. To achieve reliable detection and localisation of β-catenin, we utilised sequential quenching techniques using 1% NaBH4 and 1mM CuSO4 in 50mM ammonium acetate buffer to reduce both background and fixative-induced autofluorescence. Our results showed that sequential autofluorescence quenching is crucial in β-catenin detection, and this improved technique indicates that β-catenin is localised in the spinal cord white matter regions. Objective approach for the β-catenin localisation is highly significant as it unravelled an objective identification and illuminate the pattern of distribution of β-catenin for researcher focusing on spinal cord repair studies via the Wnt/β-catenin pathway following SCI.