A new method for the histochemical demonstration of steroid producing cells in human tissues

Summary Steroid producing cells in human tissues can be demonstrated histochemically by the use of isopropanol as substrate. The Leydig cells of the testis, the theca cells of the ovary and the zona reticularis of the adrenal are stained by this method. A possible relation of this 'secondary alcohol dehydrogenase with the enzyme that catalyses the oxidative cleavage of the cholesterol side-chain is discussed.     

A substrate-film method for the histochemical demonstration of cellulase

Summary A substrate-film method has been devized for the histochemical demonstration of cellulase. The substrate film is made of sodium carboxymethyl-cellulose, which is made insoluble in water by fixation in acid ethanol. The tissue is briefly fixed in cold formalin, washed, and sectioned with a freezing microtome. The sections are mounted on slides, and covered with a piece of carboxymethyl-cellulose film, and the slide is incubated in a warm, moist atmosphere. After incubation, the film is stained with toluidine blue, and sites of cellulase activity appear as pale or clear patches in the film.In the digestive systems of certain molluscs, cellulase has been found in the lumens of the crop and stomach, and in the lumen and absorptive cells of the digestive gland tubules. The salivary glands, and the epithelia of the crop and stomach, show no reaction.Sections of control tissue, inactivated by boiling in water, do not show any reaction.I thank the Nobel Division of Imperial Chemical Industries Ltd. for information on their product Cellofas B 10, and for permission to publish that information.     

A histochemical method for the demonstration of unsaturated lipids

Summary A method has been developed for the histochemical demonstration of unsaturated lipids in light microscopy. It is a peracetic acid-thiocarbohydrazide-silver protein sequence followed by a physical development procedure. In the present study on paraffin and cryostat sections of liver, brain and ovary, unsaturated lipids were visualized as distinct reaction products coloured various shades of brown and black. The reaction products are easier to see and the method is more efficient than the peracetic acid-Schiff method.     

A sensitive method for the histochemical demonstration of vicinal diols of carbohydrates

Summary A new sensitive method has been established for the histochemical demonstration of vicinal diols of carbohydrates in light microscopy. It consists of a periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence followed by physical development. The new method is more sensitive than the PA-TCH-SP and periodic acid-Schiff (PAS) methods employed hitherto. Its specificity is sufficient.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. III. Changes in the histochemical and chemical properties of the epithelial glycoproteins in the mucosa close to colonic tumours

Summary Histochemical, chemical and histological studies were performed on 26 specimens of human colonic tumours and 62 specimens of mucosa taken at distances of 0.5–5.0 cm from the tumour. The tumour glycoproteins were divided almost equally between three anionic types, sulphomucin, sialomucin and mixed sialomucin and sulphomucin. All showed a reduction in staining for side chainO-acylated sialic acid. In 56% of the tumours, this was accompanied by loss of glycoprotein while, in 44%, abundant mucin was still present.Histochemical examination of the mucosal specimens indicated that in 24.2% the side chainO-acylated sialic acids did not differ from normal. In 41.9% there was a focal change and in 33.9% there was a generalized field reduction in the proportion of side chainO-acyl sialic acids. The latter were subdivided into moderate and severe. Chemical analyses correlated well with the histochemical classification of the mucosal specimens and showed that, on average, the classifications focal and severe field change were not due to sampling error. Forty-five per cent of the cases showed only focal change and 40% only field change. Mucosal specimens associated with 60% of the moderately differentiated tumours showed only focal change while those associated with 75% of well-differentiated tumours showed only field change. Abnormal patterns of staining for side chainO-acylated sialic acids (a) were largely independent of the distance from the tumour, (b) occurred in the presence of a normal pattern of staining for sialomucins and sulphomucins and (c) were associated with 61.4% of the specimens that showed no discernible evidence of histological abnormality. In contrast, only one specimen showed evidence of histological change without a corresponding change inO-acylated sialic acids. The data suggest that abnormal patterns of staining forO-acylated sialic acids may represent premalignant change but their precise significance and specificity requires further studies of non-neoplastic diseases of the colon.     

A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes

The "direct coloring" thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 micron. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the detection of multiple forms of AChE in polyacrylamide or agarose gels.     

A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes

Summary The direct coloring thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 m. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the defection of multiple forms of AChE in polyacrylamide or agarose gels.In honour of Prof. P. van Duijn     

A specific histochemical method for the demonstration of the activity of pancreatic lipase

Synopsis Emulsified long-chain triglyceride, a specific substrate for the enzyme pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3), has been used in a modification of the Gomori technique for the demonstration of lipase. In the range of tissues examined (pancreas, testis, cardiac stomach and liver), true pancreatic lipase activity was revealed only in pancreatic tissue, by contrast with results obtained with less specific methods.     

A histochemical method for gross demonstration of motor end plates.

The thiocholine-ferricyanide method of Karnovsky and Roots for histochemical demonstration of cholinesterases has been applied to whole fetal and neonatal mice and chicks for the visualization of motor end plate patterns in superficial muscles or deeper muscles exposed by dissection.     

Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit

Summary Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundantO-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars withvicinal diols whose periodate oxidation is prevented by anO-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisablevicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have anO-acyl substituent located at position C-8 or C-9 (or which have two or three side chainO-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.