The demonstration of adenosinetriphosphatase activity with the electronmicroscope

Summary A new method for the demonstration of adenosine triphosphatase (ATP'ase) activity in tissue blocks with the electron-microscope has been developed. This method consists of short fixation (2–3 minutes,ph 7.3) in a OsO₄-Tris-HCl mixture, incubation with a medium containing Tris-HCl buffer, tartrate, lead and magnesium ions, ATP and sucrose (ph 6.8) and routine postfixation. The method combines satisfactory histochemical specificity with good preservation of tissue fine structure. Estimations of enzymatic activity of ATP'ase and alkaline phosphatase, as observed with the electronmicroscope, were compared with activity determinations in tissue homogenates. The occurence of a positive histochemical reaction in swollen mitochondria is discussed.With 9 Figures in the Text     

CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.     

Histochemical demonstration of the sites of activity of dehydrogenase systems with the electron microscope

In the present study a histochemical method demonstrating the activity of dehydrogenase systems was developed for electron microscopy, utilizing potassium tellurite as the hydrogen or electron acceptor. This reagent was used intravitally (intravenously, intraperitoneally, or intraluminally in hollow organs) or supravitally on small blocks of tissue for the demonstration of endogenous dehydrogenase activity. Blocks of tissue which had been frozen and thawed or which had been washed in 0.44 M sucrose to prevent endogenous activity, were used to demonstrate the activity of the succinic dehydrogenase system. In the latter case, the incubating medium contained tellurite, succinate, phosphate buffer, sucrose, and activators. The incubation was as performed either aerobically (with or without the addition of potassium cyanide) or anaerobically. The specificity and the enzymatic nature of the reactions were ascertained by appropriate control experiments. Reduced tellurite, the end product of this histochemical reaction, could be visualized in thin sections of osmium tetroxide-fixed, methacrylate-embedded tissues as crystals or fine particulate deposits of high density, localized on, or in close relationship to mitochondrial membranes. The results of these experiments are demonstrated, utilizing heart muscle (rat) as the source of the enzyme systems.     

Quantitative histochemical determination of muscle enzymes: biochemical verification

We established quantitative histochemical assays for the enzymatic activity of succinate dehydrogenase and alpha-glycerol phosphate dehydrogenase for cat skeletal muscle. A computer-enhanced image analysis system was used to quantitate the histochemical enzyme-activity reaction products. We describe a series of experiments that verify the reliability and validity of the assays. Histochemically determined enzyme activities were linear with respect to tissue thickness and reaction time. Biochemically determined enzyme activities were also linear with respect to tissue thickness and incubation time. Consecutive tissue sections, assayed either histochemically or biochemically, were used to establish a linear regression equation that allowed quantitative histochemically determined reaction rates, measured in optical density per minute, to be calibrated as nanomoles per minute.     

FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q₁₀, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.     

Microphotometric dynamic analysis of the histochemical acetylcholinesterase reaction

Dynamic analysis of the histochemical reaction of Karnovsky-Roots for acetylcholinesterase activity (AChE) is reported. Two methods were used. The first method was videography and densitometric analysis of frames from the film. The second method was direct microphotometric analysis of the reaction dynamics by the plug-method (measurement of average light transmission through a limited area of preparation or image). Special microchambers were used on the stage of an inverted microscope. The results showed the dynamics of final product accumulation in two structures of rat caudate nucleus: AChE-positive neuropil and the AChE-negative myelin bundle during histochemical incubation. Videography and densitometry of the digital images allowed morphologic and microphotometric analysis of changes in tissue sections during incubation, and the dynamic analysis permitted the study of enzyme kinetics in situ. Problems associated with microphotometric analysis of digital images for quantitative histochemical studies of the enzyme activity are discussed.     

Microphotometric dynamic analysis of the histochemical acetylcholinesterase reaction

Dynamic analysis of the histochemical reaction of Karnovsky-Roots for acetylcholinesterase activity (AChE) is reported. Two methods were used. The first method was videography and densitometric analysis of frames from the film. The second method was direct microphotometric analysis of the reaction dynamics by the plug-method (measurement of average light transmission through a limited area of preparation or image). Special microchambers were used on the stage of an inverted microscope. The results showed the dynamics of final product accumulation in two structures of rat caudate nucleus: AChE-positive neuropil and the AChE-negative myelin bundle during histochemical incubation. Videography and densitometry of the digital images allowed morphologic and microphotometric analysis of changes in tissue sections during incubation, and the dynamic analysis permitted the study of enzyme kinetics in situ. Problems associated with microphotometric analysis of digital images for quantitative histochemical studies of the enzyme activity are discussed.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of acid phosphatase in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of enzyme into the medium during incubation. Moreover fixation of the tissue sections in order to minimize enzyme diffusion and that causing a partial inactivation of the enzyme, is no longer necessary. In the histochemical system the enzyme catalyzes the hydrolyzes of naphthol AS-BI phosphoric acid. The enzyme localization is visualized by means of simultaneous coupling of the released naphthol with hexazotized pararosanilin. Problems involved in the histochemical demonstration of the enzyme are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).The author wishes to acknowledge the valuable technical assistance of Mr. E. D. J. Lindenbergh and Mr. A. H. T. Vloedman.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of lactate dehydrogenase activities in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of lactate dehydrogenase into the medium during incubation. In the histochemical system the NAD⁺-dependent enzyme catalyzes the electron transfer from lactate into NAD⁺. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Amytal is incorporated into the incubating-medium to block electron transfer to the cytochromes. Problems involved in the histochemical demonstration of lactate dehydrogenase activity are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

A Modified Freezing-Drying Apparatus for Tissues

The preparation of tissues by the freezing-drying technic is preferred for many histochemical studies because of the rapid fixation, avoidance of deleterious action of chemical fixation and extraction by aqueous and lipid solvents in fixation, dehydration and clearing; to facilitate this procedure a freezing-drying apparatus has been constructed which permits cutting of sections within five hours after the fresh tissue is obtained. Liquid nitrogen provides a most efficient moisture trap and in conjunction with a heating element provides any desired temperature down to — 80°C. during tissue drying. Paraffin infiltration is started without disassembling the equipment and completed in a few minutes. Most stains and histochemical reactions for enzyme and other substances can be applied directly to sections of frozen-dried tissue cut from the same blocks.     

Studies in lipid histochemistry

Summary 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.     

Histochemical localization of trehalase activity in dorsal flight muscle of the flesh fly Sarcophaga bullata with light and electron microscopy.

Trehalase activity in flight muscle of the flesh fly Sacrophaga bullata is detected histochemically at light- and electron-microscopic levels by using diaminobenzidine, glucose oxidase and peroxidase in the incubation medium. The association of trehalase activity with the inner mitochondrial membrane is confirmed. Biochemical assay shows that about 50% of the initial total trehalase activity is lost from the tissue during the histochemical processing and about 50% remains for histochemical detection.     

Cryostat Techniques: Methods for Improving Conservation and Sectioning of Tissue

Tissues have been conserved for satisfactory enzymatic histochemical assay for up to 12 mo by low temperature storage (in a dry ice chest or at -40° C) and by measures designed to offset the deleterious effects of sublimation of H₂O by using the following modifications of standard procedures (which include steps to give a mechanical support to otherwise fragmenting sections): 1. tissue blocks are coated with a polystyrene solution between each storage period; 2. modified bolts are used as tissue holders and types of bolt holders have been designed to fit on standard microtomes which permit manipulation of each tissue block independently of its mates on the same bolt holder, or the simultaneous cutting of all blocks on any one holder with each advance of the microtome feed; 3. tissues are coated with 20% polystyrene in methylene chloride prior to cutting and rubber cement painted on the slide as an adhesive. Lillie's 20% polystyrene diethylbenzene is used as a mounting medium. Other details of practical importance include the technique of freezing, control of moisture within a cryostat and on the microtome, and tests on histochemical procedures.     

Single motoneuron succinate dehydrogenase activity

We have developed a quantitative histochemical assay for measurement of succinate dehydrogenase (SDH) activity in single motoneurons. A computer image processing system was used to quantify the histochemical enzyme reaction product and to follow the time course of the reaction. The optimal concentration for each of the ingredients of the incubation medium for the SDH reaction was determined and the importance of using histochemical "blanks" in the determination of enzymatic activity was demonstrated. The enzymatic activity was linear with respect to reaction time and tissue thickness. The procedure described meets the criteria generally considered essential for establishment of a quantitative histochemical assay. The assay was then used to examine the SDH activity of cat and rat motoneurons. It was found that motoneurons with a small soma size had a wide range of SDH activity, whereas those with a large soma size were restricted to low SDH activity.     

Electron microscopical study of a cytosolic enzyme in unfixed cryostat sections: demonstration of glycogen phosphorylase activity in rat liver and heart tissue

Summary Glycogen phosphorylase activity has been demonstrated at the ultrastructural level in liver and heart tissue of fasted rats. Unfixed cryostat sections were incubated by mounting them on a semipermeable membrane stretched over a gelled incubation medium. The medium contained a high concentration of glucose 1-phosphate which enables indirect detection of glycogen phosphorylase activity on the basis of the synthesis of glycogen. Tissue fixation, dehydration and embedding for electron microscopical study were performed after the incubation had been completed. The ultrastructure of both liver and heart tissue was rather well preserved. Glycogen granules resulting from glycogen phosphorylase activity were found in the cytoplasmic matrix of both hepatocytes and cardiomyocytes; no relationship with membranous structures could be detected. It is concluded that the semipermeable membrane method is well suited for localizing cytosolic enzyme activities at the ultrastructural level without prior tissue fixation; this opens further perspectives for correlations between histochemical and biochemical data.     

A modified semipermeable membrane technique for carbonic anhydrase histochemistry of the nervous system

We present a modification of Hansson's method for the demonstration of carbonic anhydrase activity. Using a semipermeable membrane together with a fluid incubation medium, frozen sections of aldehyde-fixed tissue were incubated without floating or dipping. Thin sections (thickness, 20-40 microns) were mounted on the outer surface of a tubular-shaped, semipermeable cellophane dialysis membrane containing the incubation fluid. After incubation for 25-30 min at room temperature, the sections were rinsed in buffer and treated with 0.5% (NH4)2S solution. The histochemical reaction was fully inhibited by 10(-4) M acetazolamide.     

Histochemical quantification of dopa-oxidase (tyrosinase) activity in epidermal melanocytes

Summary The graphical method of Lineweaver-Burk was applied to the histochemical system to obtain quantitative information on the dopa-oxidase activity in human epidermal melanocytes. In vitro experiment showed that the incubation time necessary for yielding a certain amount of dopa-melanin is related to the amount of enzyme present. In the case of histochemical experiment, the time required for the first appearance of dopa-melanin deposition in melanocytes at various substrate concentrations was plotted against the reciprocal of the substrate concentration. This technique made it possible to compare the dopa-oxidase activity at various sites of the tissue.     

Histochemical quantification of dopa-oxidase (tyrosinase) activity in epidermal melanocytes.

The graphical method of Lineweaver-Burk was applied to the histochemical system to obtain quantitative information on the dopa-oxidase activity in human epidermal melanocytes. In vitro experiment showed that the incubation time necessary for yielding a certain amount of dopa-melanin is related to the amount of enzyme present. In the case of histochemical experiment, the time required for the first appearance of dopa-melanin deposition in melanocytes at various substrate concentrations was plotted against the reciprocal of the substrate concentration. This technique made it possible to compare the dopa-oxidase activity at various sites of the tissue.     

Histochemical technique for the demonstration of pyruvate kinase activity

We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP and D-glucose to yield D-glucose-6-phosphate and ADP. The D-glucose-6-phosphate is oxidized by exogenous and endogenous D-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP+, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Sodium azide and amytal are included to block electron transfer to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal muscle. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.