Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

Summary Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.     

Localization of Glucose-6-phosphate Dehydrogenase Activity on Ribosomes of Granular Endoplasmic Reticulum, in Peroxisomes and Peripheral Cytoplasm of Rat Liver Parenchymal Cells

Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm and in peroxisomes. Fixation may be held responsible for selective inactivation of part of glucose-6-phosphate dehydrogenase activity. In the present study, we applied the ferricyanide method for the demonstration of glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of rat liver in combination with the semipermeable membrane technique and in isolated rat liver parenchymal cells. Isolated liver parenchymal cells were permeabilized with 0.025% glutaraldehyde after NADP⁺ protection of the active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction product in sections. With the optimized method, electron dense reaction product was observed in cryostat sections on granular endoplasmic reticulum, in mitochondria and at the cell border. However, the ultrastructural morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

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.     

Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions

The extinction coefficient is essential for the conversion of cytophotometric (mean integrated) absorbance values into absolute units of enzyme activity, for instance expressed in terms of moles of substrate converted per unit time and per unit wet weight of tissue. The extinction coefficient of polymerized diaminobenzidine (polyDAB) complexed with cobalt as the final reaction product of oxidase reactions was estimated at 575 nm by comparison of the amounts of final reaction products formed after incubation of serial unfixed cryostat sections of rat kidney to demonstrate D-amino acid oxidase activity with either the tetrazolium salt method or the cerium-DAB-cobalt-hydrogen peroxide method. Both procedures resulted in similar localization patterns of final reaction product in a granular form in epithelial cells of proximal tubules in rat kidney. The granules were peroxisomes. Linear relationships were found for both methods between the specific amounts of final reaction product generated by D-amino acid oxidase activity and incubation time. The cerium salt method gave rise to 7.4 times higher absorbance values of final reaction product generated per unit time and per unit wet weight of tissue than the tetrazolium salt procedure. The extinction coefficient of tetranitro BT-formazan is 19 000 at 557 nm. Therefore, the cytophotometric extinction coefficient of the poly DAB-cobalt complex as final reaction product of oxidase reactions was established to be 140 000.     

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle

Summary Meijer's semipermeable membrane technique for acid phosphatase was modified by treating sections immediately after incubation with 70% ethanol for 30 min at room temperature instead of with formalin. The modification improved the validity of the technique considerably, by stabilising the specific final reaction product formed in presumed enzyme-containing sites. The modified technique thus seems promising for assaying the activity of acid phosphatase in sections of skeletal muscle.     

A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions.

The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between section and gelled incubation medium. The amount of final reaction product precipitated in a granular form was about four times higher with this technique in comparison with conventional procedures using fixed sections and aqueous incubation media. The specificity of the reaction was proven by the 70% reduction of the amount of final reaction product when incubating in the presence of substrate and D,L-beta-hydroxybutyrate, a specific inhibitor of D-amino acid oxidase activity. Cytophotometric analysis of liver sections revealed that the specific test minus control reaction was linear with incubation time and section thickness. The Km value of the enzyme of 10.3 +/- 2.7 mM, as determined in periportal areas, is about five times the value found with biochemical methods in liver cell homogenates. The enzyme activity in periportal areas is about five times the activity in pericentral areas. Fasting (24 and 48 hr) induced a significant decrease in D-amino acid activity in periportal and pericentral areas. The possible physiological role of the enzyme in liver is discussed.     

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.     

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver

Summary The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light-and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mm diaminobenzidine, 44 mm hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 m. Catalase in rat liver showed a K_m value of 2.0 mm for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mm. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.     

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.     

Ultrastructural localization of L-alpha-hydroxy acid oxidase in rat liver perioxisomes.

The localization of L-alpha-hydroxy acid oxidase in rat liver peroxisomes was studied using slight modifications of the Shnitka and Talibi (1971) method. Best results were obtained with formaldehyde fixation and incubation with glycolate as substrate. Following incubation the copper ferrocyanide reaction product was amplified with 3,3'-diamino-benzidine according to Hanker et al. (1972a,b). Dense reaction product was visible in hepatocyte peroxisomes by light and electron microscopy. Some diffusion of enzyme and/or reaction product into the adjacent cytoplasm occurred around the peroxisomes. Apparent non-specific deposits occurred on the plasmalemma, in the nucleus, and occasionally over mitochondria. Glutaraldehyde fixation severely inhibited enzymatic activity, and the enzyme showed less activity toward L-lactate and DL-alpha-hydroxybutyrate.     

Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

A new method for flat-embedding large native cryostat sections for targeting small preneoplastic lesions in comparative ultrastructural and ultracytochemical investigations

 Ultrastructural studies of rare and small cellular lesions in pathologically altered tissue are difficult to perform by applying conventional electron microscopic preparation. The search for lesions, often consisting of only a few cells in randomly obtained small specimen blocks, is time consuming and often without success. The methodological requirements for comparative enzyme cytochemical and morphological studies, i.e., preservation of both enzyme activity and ultrastructure, are divergent. By processing large native cryostat sections for electron microscopy, small preneoplastic focal lesions were successfully targeted in liver and kidney. Glucose-6-phosphatase, alkaline phosphatase, acid phosphatase, catalase, and cytochrome c oxidase activities were distinctly localized to endoplasmic reticulum, canalicular membrane, lysosomes, peroxisomes, and mitochondria, respectively, in the morphologically altered cells. Fixation of serial cryostat sections and enzyme reactions were both carried out through a semipermeable membrane except those for cytochrome c oxidase, which was demonstrated after fixation through the membrane by floating the section in incubation medium containing cytochrome c. Thereafter, the sections were flat embedded and polymerized between epoxy resin disks and aluminum dishes fitting exactly together. The objects of interest were identified in the light microscope, cut out, and reembedded in reversed gelatine capsules. By using this technique an ultrastructural preservation was achieved similar to that seen after immersion fixation. The enzyme activities were clearly localized without diffusion of the reaction product or unspecific deposits. The procedure permits precise targeting and complex studies of rare and small lesions, and opens new perspectives for the use of cryo-preserved tissue. Accepted: 10 March 1998     

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.     

Localization and cytophotometric analysis of cathepsin B activity in unfixed and undecalcified cryostat sections of whole rat knee joints

Cathepsin B activity is demonstrated histochemically with a post-coupling method using Z-Arg-Arg-4-methoxy-2-naphthylamide as substrate and Fast Blue BB as coupling reagent in unfixed and undecalcified cryostat sections of whole rat knee joints. Sections were attached to transparent tape to keep the integrity of the tissue intact, such attachment being essential for precise precipitation of the final reaction product at sites of enzyme activity. Also essential was inclusion of polyvinyl alcohol in the enzyme incubation medium. High cathepsin B activity was found in osteoclasts, chondrocytes, fibroblasts, synovial cells, and bone marrow cells in knee joints after induction of arthritis. The final reaction product was precipitated as fine cytoplasmic granules probably corresponding to lysosomes. The reaction was specific because addition to the incubation medium of selective inhibitors of cathepsin B-like activity completely blocked the activity. The amount of final reaction product in synovium and in bone marrow cells was analyzed cytophotometrically. Specific formation of final reaction product was linear with incubation time up to 60 min at 37 degrees C and with section thickness up to 12 microns. Variation of the substrate concentration in the incubation medium revealed a KM value of 1.86 +/- 0.36 mM in synovial cells and 2.48 +/- 0.51 mM in bone marrow cells and Vmax values (expressed as mean integrated absorbance) of 1.18 +/- 0.10 in synovial cells and 1.02 +/- 0.11 in bone marrow cells. Both KM and Vmax values were significantly different in synovial cells and bone marrow cells (p less than 0.01) which could be owing to the presence of different isoenzymes in these tissues. We conclude that the described post-coupling method is sufficient to yield precise localization and that the method is valid for quantitative purposes.     

Ultracytochemical demonstration and probable localization of 3β-hydroxysteroid dehydrogenase activity with a ferricyanide technique

Summary In order to localize 3-hydroxysteroid dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor, and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium.A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3-hydroxysteroid dehydrogenase. The addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.     

Cytochemical and biochemical demonstration of an ATPase in membranes of human peroxisomes.

We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.     

A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme

Summary A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH 8.0, 0.5 mm inosine, 0.47 mm methoxyphenazine methosulphate and 1 mm Tetranitro BT. An enzyme film consisting of xanthine oxidase was brought onto the object slides before the section was allowed to adhere. The specificity of the reaction was proven by the low amount of final reaction product generated when incubating in the absence of inosine. Moreover, 1 mm p-chloromercuribenzoic acid, a non-specific inhibitor of purine nucleoside phosphorylase, inhibited the specific reaction by 90%. The specific reaction defined as the test reaction, in the presence of substrate, minus the control reaction, in the absence of substrate was linear with incubation time at least up to 30 min as measured cytophotometrically. A high activity was observed in endothelial cells and Kupffer cells of rat liver and a lower activity in liver parenchymal cells. Pericentral hepatocytes showed an activity higher than that of periportal hepatocytes. In human liver, purine nucleoside phosphorylase activity was also high in endothelial cells and Kupffer cells, but the activity in liver parenchymal cells was only slightly lower than it was in non-parenchymal cells. The localization of the enzyme is in agreement with earlier ultrastructural findings using fixed liver tissue and the lead salt procedure.     

Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.     

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle

Summary The reproducibility, specificity and validity of Meijer's semipermeable membrane simultaneous coupling technique for the assay of acid phosphatase activity in sections of skeletal muscle have been investigated quantitatively, using naphthol AS-BI phosphate as the substrate and hexazotised Pararosanaline (HPRA) as the coupler. With this technique, unlike conventional techniques, presumed specific final reaction product (FRP) is evident in three different histological sites in normal skeletal muscle; first, as intensely coloured red granules within muscle fibres; second, as a diffuse reddish colouration throughout the sarcoplasm of all muscle fibres (the intrafibre areas); and third, in certain connective tissue elements between the muscle fibres (the interfibre areas). The mean absorbance of the FRP (at its absorption maximum, 530 nm) formed in each of these sites after a constant incubation time does not differ significantly in serial sections. 6 mM sodium molybdate, an acid phosphatase inhibitor, reduces the mean absorbance by 50% in the intrafibre areas, but in the interfibre connective tissue areas, 1 mM is sufficient. In contrast, 10 mM EDTA, an alkaline phosphatase inhibitor, has a negligible effect on the formation of specific FRP. Thus, Meijer's technique appears to be reproducible and specific. The mean absorbance of the FRP formed in each of the three reactive histological areas increases linearly with incubation time and section thickness. The maximum amount of FRP is formed at pH 5 and when the substrate concentration is above about 4 mM. However, some of the FRP in the intrafibre areas is unspecific, and arises from the transformation of adsorbed HPRA to a purple-coloured product having an absorption maximum at 570 nm. Much of the non-specific FRP appears after the incubation has been terminated with formalin, and reaches a maximum several hours after the sections have been subsequently mounted. As a consequence, Meijer's technique is not entirely valid.