A gel-sandwich technique for the qualitative and quantitative determination of dehydrogenases in the enzyme histochemistry. I. Development of the new methods on the example of LDH (E.C. 1.1.1.27).

Abstract

A gel-sandwich technique for the histochemical demonstration of dehydrogenase is introduced with LDH set up as an example. Especially suitable, of the gels examined, for this technique is 1.5% W/V agar-agar low gel strength. In it several reaction ingredients for the histochemical reaction are dissolved. Considering LDH the following gel composition showed good results: 1.5% W/V agar-agar low gel strength, 5 mM TNBT in 150 microliter DMF, 120 mM L-lactate, 3--5 mM NAD+, 10 mM amytal, 22,4--32 X 10(-5) M Meldola Blue, 160 mM soldium phosphate buffer pH 7.6 (total solution of 1 ml). After the solidification of the gel, gel-bars were frozen with CO2-snow. The 40--80 micrometer thick gel slices were gained in the cryostat. Of the three different arrangement possibilities of the gel slices and the tissue-sections a sandwich arrangement (cover-gel slice--tissue section--ground-gel slice) produced the best results. The enzyme reaction is started by thawing of the gel slices (together with the tissue sections) and by putting them between the hotplate and the evaporator-head-piece, especially developed for this technique. The gel slices also remain in combination with the tissue sections after the reaction. The influence of the gel in combination with the electron carrier Meldola Blue on the spontaneous reduction rates of ditetrazolium salts in day light, were examined as well as the diffusion rates of TNBT and NADH out of gel slices and the influence of DMF and DMSO on the LDH activity. This technique prevents both, the loss of enzymes and the loss of reduction equivalents. There are given presuppositions for qualitative and quantitative histochemical investigations as well. The advantages of the new gel technique are discussed.