Simultaneous histochemical assay of two dehydrogenases in the same cell.

摘要: A new approach has been developed for the simultaneous assay of activities two enzymes (lactate and succinate dehydrogenases) in same cell sections unfixed liver. The sections, mounted on coverslips, were placed top 0.6-mm thick 0.8% low gelling-temperature agarose films containing substrates both (70 mM lactate 50 succinate, respectively) plus 80 Tris-HCl buffer (pH 7.5), 5 EDTA, 10 NaN3, 1.5 NAD+, 1.2 Nitro BT 0.26 phenazine methosulphate. integrated absorbance (A) at 585 nm final reaction product formazans deposited by a selected hepatocyte was measured continuously 37 degrees C as function incubation time, using Vickers M85 microdensitometer. intercept A0 A-axis linear regression line time determined. After known t, A1, noted section another gel film lacking order to estimate either formed or lost from cell. A2 remeasured. velocities (activities) vL vS dehydrogenases, respectively, calculated following equations: = [(A1-A2) - A0(1- alpha L)]/(1-alpha L)t (A2-alpha LA1)/(1-alpha where L A2/A1 hepatocytes incubated only substrate. This parameter found be virtually constant (0.44) over wide range vL.(ABSTRACT TRUNCATED AT 250 WORDS)