A comparison between the one-step and the two-step copper thiocholine procedure for the cytochemical localization of cholinesterases

Summary A comparison between the one-step and the two-step copper thiocholine procedure for the subcellular localization of cholinesterases was under-taken. The results indicate that only under experimental conditions with short incubation times and precise control of the conversion into sulphide is the localization of the primary precipitate and that of the secondary precipitate identical. It was concluded that the conversion of the primary precipitate into Cu-sulphide is not necessary and can lead to artefacts.     

Electron microscopic demonstration of cholinesterases in nervous tissue

Summary Acetylcholinesterase was demonstrated at ultrastructural level in the motor nerve cells of rat's spinal cord using the Karnovsky-Roots modification of Koelle's thiocholine method. Selective inhibitors were employed to check the validity of the reaction.Prolonged formaldehyde fixation improved the poor penetration of the reactive agents and diminished the relatively large crystal size of the end product, which were the two main difficulties of the method. The preservation of ultrastructure was highly improved, when thin sections were made without freezing using a tissue chopper.Acetylcholinesterase was localized in the nuclear envelope, in the rough-surfaced endoplasmic reticulum, in medium-sized vesicles of the Golgi apparatus, and around synaptic terminals. Synaptic vesicles were found negative.     

The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases

Summary The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.     

The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases.

The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.     

A method for the fine-structural demonstration of lectin receptors

A new two-step method using an Fc-fragment/ferritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

A method for the fine-structural demonstration of lectin receptors

Summary A new two-step method using an Fc-fragment/férritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

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.