Minimizing shrinkage of acute brain slices using metal spacers during histological embedding

The morphological structure of neurons provides the basis for their functions and is a major focus of contemporary neuroscience studies. Intracellular staining of single cells in acute slices is a well-established approach, offering high-resolution information on neuronal morphology, complementing their physiology. Despite major technical advances, however, a common histological artifact often precludes precise morphological analysis: shrinkage of the sampled tissue after embedding for microscopy. Here, we describe a new approach using a metal spacer, sandwiched between two coverslips to reduce shrinkage of whole-mount slice preparations during embedding with aqueous mounting medium under a coverslip. This approach additionally allows imaging the slices from both sides to obtain better quality images of deeper structures. We demonstrate that the use of this spacer system can efficiently and stably reduce the shrinkage of slices, whereas conventional embedding methods without spacer or with agar spacer cause severe, progressive shrinkage after embedding. We further show that the shrinkage of slices is not uniform and artifacts in morphology and anatomical parameters produced cannot be compensated using linear correction algorithms. Our study, thus, emphasizes the importance of preventing the deformation of slice preparations and offers an effective means for reducing shrinkage and associated artifacts during embedding.