Osteoclast visualization: Tartrate-resistant acid phosphatase activity staining using NewFuchsin compatible with non-aqueous mounting and tissue clearing.

Tartrate-resistant acid phosphatase (TRAP) staining is widely used to stain osteoclasts in histological bone sections. The red dye formed by the conventional TRAP enzymatic reaction using naphthol AS-MX (or AS-BI) phosphate and fast red-violet (or garnet) chromogens is readily soluble in alcohol or xylene and requires air-drying prior to cover slipping or the use of an aqueous mounting medium. However, the use of an aqueous mounting medium makes it difficult to store stained specimens for a long time. In this modified method, a new fuchsin (NewFuchsin) was used as a chromogen, which enabled dehydration and clearing after staining and the use of a non-aqueous organic solvent-based mounting medium. Samples prepared using this modified TRAP activity staining method (NewFuchsin TRAP staining) have the following advantages over conventional TRAP staining:•The staining of sections provides a clear histological image and allows for long-term preservation.•The red dye formed by NewFuchsin TRAP staining can be detected not only in the bright field, but also in the fluorescent field.•Combined with tissue clearing using ethyl cinnamate, osteoclasts are observed using three-dimensional imaging.