a tissue preparation on a slide a blot or dot on a filter membrane a dot on plastic is dipped into the indoxyl tetrazolium solution for a period of 1 30 minutes prefereably 5 20 minutes at 19 u00b0 37 u00b0 c preferably at room temperature 20 u00b0 30 u00b0 c the color develops and precipitates spontaneously at the location of the enzyme reaction is stopped by washing the object in water and air drying step 1 0 2 gr of 5 bromo 4 chloro 3 indolyl phophate toluidine salt bcip obtainable from sigma chemical co st louis mo usa is dissolved in 4 ml dimethyl formamide dmf step 2 the solution obtained in step 1 is added to 1 liter 0 1m tris hcl ph 9 5 containing 5 mm of magnesium chloride followed by rigorous stirring for about 10 minutes step 3 300 mg of nitro blue tetrazolium chloride nbt also obtainable from sigma chemicals are dissolved in 4 ml of 70 dmf and added to 1 liter of the bcip solution obtained in step 2 followed immediately by vigorous stirring store cold and dark example 2 use of the bcip nbt mixture in a diagnostic assay 1.