The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique

Summary In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD⁺, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

A one-step cobalt-ferrocyanide method for histochemical demonstration of acetylcholinesterase activity in central nervous system tissue.

We introduce a one-step histochemical method with cobalt as the precipitating agent for ferrocyanide for the light microscopic demonstration of acetylcholinesterase activity. This method was used to demonstrate acetylcholinesterase in normal cortical fibers and neurons, as well as pathological elements such as plaques and tangles. This procedure can also be easily combined with immunohistochemical methods that use diaminobenzidine as a chromogen.     

Improvement of the method of Karnovsky and Roots for the histochemical demonstration of acetylcholinesterase

Summary The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain, a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

Improvement of the method of Karnovsky and Roots for the histochemical demonstration of acetylcholinesterase

The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures.     

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.     

The histochemical demonstration of choline acetyltransferase by semipermeable membrane technique

Summary The application of the semipermeable membrane technique in light microscopical demonstration of choline acetyltransferase is described. The method founds upon earlier developed lead salt techniques. Use of semipermeable membranes fully prevents any loss of enzyme by dissolvement or inactivation during fixation. Addition of NaCl to the incubation medium markedly increases the activity of choline acetyltransferase.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

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.     

Improved method for the histochemical demonstration of glucose-6-phosphatase activity

Summary Methodological studies on the histochemical technique for the demonstration of G6Pase activity showed that the occurrence of common artifacts: morphological destruction, extracellular precipitation of reaction product and nuclear staining are dependent on the concentration of lead nitrate, buffer and substrate. By studying the effects of systematic variation of the incubation media on the histochemical reaction optimal concentrations of either of these components were determined. An improved medium containing 3.6 mM lead nitrate, 40 mM tris-maleate buffer, pH 6.5, 10 mM G6P and 300 mM sucrose was used for the study of G6Pase distribution patterns in liver acini of juvenile and adult rats of both sexes and in those of starved adult female rats. The results obtained indicate sex dependent differences in the functional organization of the liver acinus and furthermore demonstrate the rapid functional adaptability of liver parenchyma to changes of the nutritional situation.Supported by a grant of the Deutsche Forschungsgemeinschaft     

Improved method for the histochemical demonstration of glucose-6-phosphatase activity.

Methodological studies on the histochemical technique for the demonstration of G6Pase activity showed that the occurrence of common artifacts: morphological destruction, extracellular precipitation of reaction product and nuclear staining are dependent on the concentration of lead nitrate, buffer and substrate. By studying the effects of systematic variation of the incubation media on the histochemical reaction optimal concentrations of either of these components were determined. An improved medium containing 3.6 mM lead nitrate, 40 mM tris-maleate buffer, pH 6.5, 10 mM G6P and 300 mM sucrose was used for the study of G6Pase distribution patterns in liver acini of juvenile and adult rats of both sexes and in those of starved adult female rats. The results obtained indicate sex dependent differences in the functional organization of the liver acinus and furthermore demonstrate the rapid functional adaptability of liver parenchyma to changes of the nutritional situation.     

New method for the histochemical demonstration of dipeptidyl aminopeptidase I activity using a novel anthraquinoyl hydrazide substrate.

A new method for the histochemical localization of dipeptidyl aminopeptidase I (DPP I, cathepsin C), based on a newly synthesized substrate-Gly-L-Phe-5-chloro-1-anthraquinoyl hydrazide.HCl (Gly-Phe-CAH), is proposed. The enzyme activity liberates 5-chloro-1-anthraquinoyl hydrazine (CAH)--a water-insoluble brown-reddish compound, which precipitates on the enzyme locations. The primary reaction product reacts simultaneously or, otherwise, by post-coupling with p-anisaldehyde (p-AA), thus converting to the reddish-violet amorphous hydrazone--the final reaction product. The validity of enzyme localization is thus assured by the insolubility of the primary reaction product and does not depend on the rate of the second reaction step. The enzyme studied is successfully localized in different rat organs using the newly proposed technique.     

A combined myosin ATPase and acetylcholinesterase histochemical method for the demonstration of fibre types and their innervation pattern in skeletal muscle

An improved, combined staining method for myofibrillar ATPase (m-ATPase) and for acetylcholinesterase activity is described. This method allows the observations, on the same slide, of the classical histochemical m-ATPase profile following the Brooke and Kaiser technique and the neuromuscular junction morphology. Thus the pattern of innervation, nerve ending structure and number of nerve endings along the fibres is shown simultaneously for the basic differentiation between slow and fast fibres. The use of acidic and alkaline preincubation allows better visualization of endplate morphology and avoids the masking effect of a positive m-ATPase reaction. The technique has been validated on skeletal muscles from avian and mammalian species.     

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.     

Optimal parameters for the histochemical demonstration of acetylcholinesterase in plastic sections of rat brain

A modified method for improved preservation and optical resolution of acetylcholinesterase (AChE)-containing structures in adult rat brain is described. Optimal tissue preparation included fixation in paraformaldehyde 4%, glutaraldehyde 0.1%, and sucrose 7% in 0.1M Sorensen's phosphate buffer, pH 7.4, rinsing in buffer 50 mM with respect to NH4Cl and 2% with respect to sucrose, acetone dehydration, vacuum infiltration with LKB Historesin, and polymerization at 4 C, overnight incubation of 10 microns sections at 37 C in the AChE histochemical reaction mixture and silver intensification according to Hedreen et al. Demonstration of AChE enzyme activity in the cholinergic projection from the rat basal forebrain to the ipsilateral hippocampus exemplifies the usefulness of the technique. The method provides an excellent demonstration of AChE-positive axonal processes and enables the pharmacohistochemical visualization of cholinergic neurons. This procedure offers a convenient method for analysis of cholinergic neurons that avoids potential artifacts inherent in other AChE histochemical procedures.     

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.