QUANTITATIVE ELUTION OF NITRO-BLUE FORMAZAN FROM TISSUE SECTIONS

1. Various dehydrogenases can be demonstrated in tissue sections by the use of tetrazolium salts as hydrogen acceptors. These substances become reduced to insoluble formazans which are deposited in the section. 2. A solvent system for the quantitative elution of nitro-blue formazan is described. 3. The solvent is alkaline dimethylformamide (DMF); the alkalinity is achieved by the addition of 10% (v/v) of a buffer, pH about 12, to the DMF. 4. The piece of glass containing the section is cut out of the slide, placed into a test tube, and the DMF is added, followed by the buffer. The usual volumes are 0.9 ml DMF +0.1 ml buffer, but these can be altered as necessary. The formazan first turns green, and is then eluted into the DMF. Elution is usually complete within a few minutes, but may be assisted by placing the tubes into a water-bath at 40-50° C. 5. The colour is measured in a spectrophotometer at 715 mμ. It is stable for about 40 minutes. Serial dilutions are linear up to an optical density of at least 0.850. 6. The method records a linear production of formazan with respect to the amount of tissue (enzyme) present, and also with respect to the time of incubation. 7. With each test, it is necessary to incubate a control section in medium lacking both substrate and co-enzyme. This permits a correction to be made for the effects of any endogenous substrates, and also for the possibility that additional formazan is produced during the elution due to binding of the tetrazole to the tissue. 8. An optical density at 715 mμ of 0.100 in 1.0 ml solvent corresponds to 0.940 μg formazan in the sample. 9. When tested with nitro-blue tetrazolium, the optimal activity of both pentose shunt dehydrogenases occurred at very similar pH values to those found with neotetrazolium, although about three times the actual activity (in terms of hydrogen) was recorded with the former than with the latter tetrazole. © 1969 Springer-Verlag.