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Concanavalin A (ConA) binding to lipopigments (LPs) of the lipofuscin type was proved to be due to the high content of mannose. The nature of the mannose bearing compound was twofold. One part was soluble in modified chloroform-methanol-water mixture (10:10:3) corresponding possibly to the oligosaccharyl diphosphodolichol (oligo-PP-Dol) described to be increased in LPs especially of inherited types. The second part, most probably a glycoprotein (GP), was entirely resistant to various extraction procedures. The ratio of the two components varied. The deposition of the typical lipofuscin (age pigment) was dominated by the GP component. Its amount was greatest in neurolipofuscin (especially in the olivary nucleus) and in the myocardium but very little in hepatocytic lipofuscin. In human neuronal ceroid lipofuscinoses (of early juvenile, and juvenile types) both components were found in large quantities in the storage granules of the affected neurons. The “protein type variant” of the storage material (Elleder 1978) displayed the highest degree of lipid-bound mannose accumulation, the GP component being extremely low or entirely absent. In the late infantile, infantile and Kufs variants studied in paraffin sections only, the GP component was detetable, too as in the case of the secondary neuronal LP in mucopolysaccharidoses and gangliosidoses. In the dog model of NCL lipid bound mannose clearly predominated, the GP component being concentrated in the cytoplasm and on the periphery od some storage granules. The nature of the GP component, a new finding of LP analysis, is discussed. The metabolic relationship between the two components is uncertain. Neither could be identified as the component resposible for autofluorescence. However, both are most probably responsible for PAS positivity of lipofuscins. The ceroid-type histiocytic LP did not display any detectable increase in any of the mannose bearing components.

A new procedure for the detection of apolar lipids is described. It is a modification of the OTAN method (Adams, 1959) using periodic acid which oxidatively removes lower osmium derivatives from polar sites only, leaving those in apolar lipids intact and demonstrable with a-naphthylamine. Control steps for the exclusion of the possible inter-ference of some less polar complex lipids and of lipopigments are described. The described technic is superior to the conventionally used sudan dyes due partly to the fact that only aqueous solutions are employed thus excluding any extraction of lipids, partly to the more distinct coloration.

Summary: A 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.

The suitability of performic and peracetic-Schiff (PAAS and PFAS) reactions in lipid histochemistry was evaluated in comparative studies in sections and chromatograms. It was shown that the positive color obtained with PAAS and PFAS reactions in lipids is due to the plasmal reaction. In the interactions of peracids with double bonds no Schiff positive groups arise. Vic-glycol groups are formed in amounts which are dependent on the type of peracid used. It is concluded that PAAS and PFAS reactions are useless in lipid histochemistry.

It was shown by experiments with staining reactions performed “in situ” in blood smears of adults and children, in sections prepared by various procedures, in spot tests, and by chromatographic examination of extracted lipids that haemoglobin is responsible entirely for the staining of red blood cells in the acid haematein test and for the greater part for the staining in the OTAN reaction. The acid haematein test is from the point of view of lipid histochemistry neither specific nor sensitive. On the other hand the OTAN reaction is sensitive although it is not specific. It is concluded that the extraction test with chloroform-methanol must always be performed when the lipidic nature of the demonstrated substance(s) is to be proved unequivocally and that the non-lipidic nature of the residual staining must be always considered.

The 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.

Conditions for a selective extraction of nonpolar lipids from tissue sections with acetone were investigated using methods of lipid chromatography, histochemistry and quantitative determination of lipid phosphorus.

A new, simple, rapid and selective method for the histochemical detection of phospholipids is described. Pairs of sections (cold microtome, frozen and even paraffin sections) one of which is preextracted with a mixture of chloroform-methanol are stained for 6–8 minutes in a modified stabilized solution of ferric haematoxylin after Weigert and Lillie in which the aqueous solution of haematoxylin is substituted for the alcoholic one. Preparations can be mounted in aqueous as well as nonaqueous media. The presence of phospholipids is assessed from differences of the coloration between nonextracted and extracted sections. Although phospholipids are the only lipid material reacting positively with ferric haematoxylin, better results are obtained in sections from which nonpolar lipids were removed with acetone. The role of bound phosphate group in the reaction of ferric haematoxylin with lipidic (and probably also with nonlipidic substances) is briefly discussed.

A considerable portion of polar lipids survives the routine dehydration procedure for paraffin embedding with ethanol, acetone and xylene and can be detected in dehydrated blocks of tissue. Sphingomyelin, cerebrosides, sulphatides and gangliosides can be demonstrated with appropriate histochemical methods and chromatographically even in ordinary paraffin sections especially when the amount of these lipids in tissues is sufficiently high, e.g. in lipidoses and in normal myelin. In blocks of tissue dehydrated with acetone and cleared with benzen a considerably higher amount of polar lipids is present. Factors governing the preservation of polar lipids in paraffin sections are discussed.

The specifity of Holczinger's method for fatty acids was reinvestigated. It was shown that phospholipids, lipopigment and even nonlipidic substances can interfere in the reaction. To exclude such interference the reaction should be carried out also in parallel sections pretreated with hydrochloric acid one of which is extracted with anhydrous acetone. Only substances positive in section pretreated with HCl and negative after pretreatment with HCl followed by acetone extraction can be considered as fatty acids.

The effect of bromination (carried out especially with potassium tribromide and with bromine water) on various chemical groups in lipids and nonlipidic substances in tissue sections and in spot tests was investigated. Bromination blocks rapidly and effectively the reaction of double bonds with osmium tetroxide in unsaturated fatty acids, in phosphoglycerides, and nonpolar lipids, whereas in sphingolipids some residual staining persists even when the blocking period is prolonged. Similar results were obtained in the case of the UV-Schiff reaction. The vinylether bond in plasmalogens is blocked completely but not so rapidly.

In sections of human scalp and rat tongue it was shown that formaldehyde is bound to SH groups and can be demonstrated by the use of sublimate-Schiff mixture or to a lesser degree by Schiff's reagent alone. It was shown that the reaction with bound formaldehyde must be considered in the plasmal reaction when sublimate-Schiff mixture is used as a detecting agent. Further the bound formaldehyde interferes in the UV-Schiff reaction. There is no interference in the PAS reaction and in the plasmal reaction carried out with the separate application of sublimate and Schiff's reagent. The mechanism of the SH-bound formaldehyde-Schiff reaction is discussed.

The control reaction with Schiff's reagent demonstrates besides free aldehydes also plasmalogens and is not a proper control test in lipid histochemistry in cases when reactions with Schiff's reagent are used in tissues containing plasmalogens. For the elimination of genuine aldehydes neutral blocking tests should be applied whenever plasmalogens are present.

A 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 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. Tschugajev's 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, a-naphthylamine, ß-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.

The 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.

OTAN, controlled chromation—acid haematoxylin and ferric haematoxylin methods were evaluated for their capability of the demonstration of sphingomyelin in tissue sections from which phosphoglycerides were removed by alkaline hydrolysis. The OTAN method is seriously invalidated due to its strong staining of cerebrosides and sulphatides. These sphingolipids are alkali—resistent and are stained together with sphingomyelin after alkaline hydrolysis. Controlled chromation—acid haematoxylin is more suitable. It stains sphingomyelin well and cerebrosides only faintly. This method is not to sensitive, however. The ferric haematoxylin method fulfills all criteria necessary for the demonstration of sphingomyelin and is the method of choice for its demonstration “in situ”.

The 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.

The influence of different terminal rinsing bathes on the results of methods using Schiff's reagent (S.r.) was studied in tissue sections and in chromatograms. The compound resulting in the reaction of aldehydic groups with S.r. is not stable in the acidic pH range. The amount of aldehydic groups which are set free increases with the acidity of the rinsing bath. This results in a substantial weakening of the original coloration. The use of a slightly acid bisulphite-HCl bath followed by thorough washing in tap water proved to be very good in practice. Strongly acidic bathes (2 N-3 N HCl) must be avoided.