The demonstration of arylsulfatases with 4-nitro-1,2-benzenediol mono(hydrogen sulfate) by the formation of osmium blacks at the sites of copper capture.

A new method is described for the direct cytochemical demonstration of lysosomal arylsulfatases utilizing a synthetic substrate, 4-nitro-1,2-benzenediol mono(hydrogen sulfate), and a copper capture reaction. A small amount of Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6-7 H2O) formed at the subcellular sites of copper capture is then utilized as a heterogeneous catalyst to effect the oxidative polymerization of 3,3'-diaminobenzidine which results in the formation of an insoluble, highly colored osmiophilic indamine polymer at the sites of enzymatic activity. The reaction product even at this stage prior to osmication is highly visible. It is readily seen with a light microscope in 50 mum sections of fixed tissues prepared with a mechanical chopper or in 10 micron cryostat sections treated for arylsulfatase activity. Upon osmication, an electron-opaque osmium black is formed which is much less soluble than the products of either the lead or barium capture reactions currently used for the demonstration of arylsulfatase with the electron microscope. The selection of areas of plastic-embedded tissues for ultrathin sectioning is facilitated by the ready visibility of these osmium black end products on 1-2 mum plastic sections which can be studied with the light microscope. This method gives permanent specimens demonstrating arylsulfatases A or B in lysosomes and autophagic vacuoles. In addition, enzyme activity is seen occasionally in the Golgi region or lamellae of certain cells believed to be elaborating sulfated products. In these instances, it may be demonstrating sulfotransferase activity.