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The alteration of extinctions of both FITC isomers by coupling to bovine serum albumin, human, and rabbit-gamma-globulin were studied. The extinction coefficients of these FTC conjugates were established. The emission characteristics of the FITC isomers and their conjugates were investigated under conditions normaly used in fluorescence microscopy. FITC I was found as the most suitable marker for human- and rabbit-gamma-globulin. In contrast the fluorescence of FTC II-BSA conjugates is stronger than that of the corresponding FTC I-BSA conjugate.

The efficiency of the membrane methods of Meijer (1972) and Lojda (1973) in histochemical lysosome tracing was studied and compared with the results of a preparation technique developed by us, employing fresh-frozen celloidin-coated sections; in addition, these methods were compared with results obtained with conventional formalin-sucrose-fixed livers. The lysosomes were traced by reactions for ACPase, beta-glucuronidase, arylesterase and acid-beta-galactosidase activity in rat livers systematically harvested at different time points of occurence of their maximal and minimal activities during the 24-h period, thus indicating a heterogeneity of lysosomes. 3. Optimal results with respect to the morphological appearance of lysosomes were obtained in fresh-frozen celloidin coated sections. This preparation method also caused no enzyme inhibition or loss, thus delivering comparatively the strongest reactions in livers and in enzyme models. 4. Day-time-dependent extralysosomal enzyme activities regularly occur in hepatocytes. Extralysosomal localizations however, are not a consequence of technically induced enzyme diffusion; they are best visualized in celloidin-coated sections; the membrane method produces less satisfactory results, and formalin-sucrose-fixed livers were least satisfactory.

The influence of the mode of polymerization of GMA on the enzyme activities of differently fixed rat livers was studied. It was found that with increasing time of UV-polymerization several enzyme activities were inhibited whereas chemical polymerization was comparatively less harmful. The histochemical reactivity of substrates such as glycogen was not impaired by the mode of polymerization, which is in contrast to the increased basophilia of DNA and RNA after UV-polymerization.

The use of gaseous (DEPC, BOV) postfixation of frozen-dried tissues for the fixation of non-hormone proteins was systematically studied. The fixation ability was tested on the "protein droplet model" i.e. the protein droplets formed in mouse liver after intravenous injection of bovine serum albumin (BSA). 1. There is no difference in the fixation ability for BSA between DEPC and BQV. 2. Both fixatives yield a very poor fixation of BSA in frozen-dried livers. In consequence, this protein is already extracted during section mounting and incubation for immunofluorescent tracing causing a strong substantial loss. 3. Remaining protein traces, however, are still immunoreactive indicating that DEPC and BQV fixation does not affect the antigenicity in considerable extent.

The influences of paraffin and GMA-embedding on acid phosphatase, esterase and beta-glucuronidase activity of differently fixed or freeze substituted rat livers were studied. 1. Embedding generally causes a reduction of the enzyme activities but improves considerably the quality of the microscopical pictures when compared with appropriate cryostat sections. Embedding therefore may serve as a very useful tool for detail studies on the cytological level. 2. The embedding media act differently on the reactive sites: a. Paraffin causes a heavy denaturation of the enzyme activity in lysosomes but preserves the activities of the ergastoplasmic (= "microsomal") enzymes. The degree of denaturation increases with increasing embedding temperature. b. GMA-embedding delivered opposite effects by preserving lysosomal activities and quenching endoplasmic enzymes. UV-polymerization of GMA causes a general inactivation of enzymes. 3. The histochemical reactivity of substrates such as glycogen was not influenced by the embedding. However, its most natural localization is achieved by freeze drying or isopropanol freeze substitution followed by GMA-embedding.