M. Elleder

Remarks on the "OTAN" Reaction

SummaryA series of experiments with the standard OTAN reaction and its modifications was performed in human and animal tissue sections. The coloring obtained in the original OTAN reaction cannot be considered as unequivocal characterisation for hydrophobic and hydrophilic lipids because both kinds of lipids can produce a whole scale of colors ranging from light red to black.The black coloring of hydrophobic lipids can develop after application of α-naphtylamine which does not react only by forming a complex but also in the sense of oxidation-reduction.The red coloring of hydrophobic lipids resistant to prehydrolysis by 1 N NaOH in atheromatous plaques and in arcus senilis corneae could be considered erroneously as presence of the sphingomyelin.Hydrophilic lipids can be stained already after 10 minutes treatment with osmium tetroxide and potassium chlorate mixture. After application of α-naphtylamine a dark brown to brown-black color can develop in some cases which does not signify the presence of hydrophobic lipids. Nonlipid substances can be stained in the OTAN method as well.Modifications of the OTAN method enabling the differentiation of hydrophobic and hydrophilic lipids and lipopigment are described. These consist of pre-extraction with cold acetone or chloroform-methanol or oxidation by periodic acid.

Remarks on the detection of osmium derivatives in tissue sections

SummaryThe reaction of substances with osmium tetroxide in tissue sections is evident when primary or secondary blackening occurs. Many substances are not colored, however, despite the fact that they may react with osmium tetroxide. Osmium derivatives bound in such structures may be either in an insufficient amount or in a higher valency state or both. For the demonstration of such weakly colored bound osmium derivatives some chemical reagents including those used in analytical chemistry were tested. Not all reagents used were equally effective. Tschugajevs reagent with thiourea is unreliable for “in situ” detection in tissue sections because the resulting colored complex is soluble and escapes from the section into solution. Yellow ammonium sulphide is not sensitive enough. N-phenyl-p-phenylene diamine, N-dimethyl-p-phenylene diamine, benzidine, o-dianisidine, N-diethyl-p-phenylene diamine sulphate, p-aminophenol, p-phenylene diamine, N-methyl-p-aminophenol sulphate, 2,4-diaminophenol hydrochloride, α-naphthylamine, β-naphthylamine, diphenylcarbazone, 8-amino-1-naphthol-3,6-disulphonic acid (H acid), aniline, tannin, proved to be the most suitable detecting reagents. By these reagents large quantities of “osmium pigments” could be demonstrated in many structures even after the most efficient lipid extraction methods.

Demonstration of Enzymes in Cells Cultured on Semipermeable Membrane in a Double Chamber

SummaryThe description is presented of a simple device for short-term cell culturing on semipermeable membrane for the histochemical demonstration of total enzyme activities. The results are given of lysosomal enzyme detection in HeLa cells.

Prolonged methanol fixation of soluble mucosubstances in mucopolysaccharidoses

SummaryA simple and efficient method for the demonstration of highly water soluble acid mucosubstances in cold microtome sections is described. It consists of prolonged treatment of cold microtome sections with methanol (for at least 1 h) and subsequent staining with 0.1% azure A in distilled water or in 30% methanol. The procedure is recommended particularly for the bioptical examination of mucopolysaccharidoses.

Remarks on the cis-aconitic anhydride method

SummaryThe reliability of cis-aconitic anhydride (CAZA) method in the histochemical detection of phospholipids was investigated. It was found, that the prescribed oxidative polymerization with cobalt chloride and sodium periodate of fixed sections is rather ineffective because the bulk of phosphlipids was detected in pooled solvents used in preparative steps. Even if residual phospholipids are revealed in sections the method is not recommended in practical histochemistry.

Reappraisal of osmium tetroxide and OTAN histochemical reactions

ConclusionsThe paper “Reappraisal of osmium tetroxide and OTAN histochemical reactions” does not disprove our conclusions presented in our previous papers [Histochemie 13, 276–282 (1968); 14, 47-64 (1968)]. We must state again that the resulting color in the OTAN reaction is not an unequivocal criterion for distinguishing between hydrophilic polar lipids and hydrophobic non polar lipids. A distinction is possible using supplementary reactions recommended in our previous paper [Histochemie 14, 47–64 (1968)]. The reaction of non lipidic substances cannot be disregarded.