Selection of a simple protease procedure for identifying mast cells in routinely processed human tissues

Proteases present in mast cell granules have been harnessed to demonstrate mast cells in human tissues. A number of substrate mixtures were tested. D-Val-Leu-Arg-4-methoxy-2-naphthylamide (MNA) plus Fast Blue B was the best for identifying human mast cells, yielding the most specific and complete staining. The procedure is simple and the results are permanent. Cryostat sections of aldehyde-fixed routine preparations or paraffin sections of Carnoy-fixed tissues give the most satisfactory results. Mast cells are stained a strong red color that stands out distinctly from the surrounding tissues, so that they can be easily identified by simple microscopy. A double-staining technique, first for protease and subsequently using Alcian Blue, showed that as progressive protease staining occurs, the alcianophilia of mast cells is lost. This procedure demonstrated that mast cells in the mucosa of human gut generally required longer incubations to develop protease staining than in other connective tissue sites. In post-mortem tissues, mast cells retain their protease activity well and so can be demonstrated in cryostat sections of aldehyde-fixed material, giving a more complete picture than with Alcian Blue. The synthetic substrate D-Val-Leu-Arg-MNA can be recommended for routine identification of mast cells in human tissues.