Unfixed Tissue Sections Research Articles

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5 micron thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD+ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500 mumol/l) of co-factor employed. The relationship between NAD+ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD+ concentration was greater than 250 mumol/l. The apparent Michaelis-Menten constant (Km) was 29 mumol/l and Vmax 0.63 mumol NAD+ reduced/min/cm3 corpora lutea.

Light microscopic autoradiographic localization of [ 3H]oxytocin binding sites in the rat brain, pituitary and mammary gland

An autoradiographical oxytocin (OXT) labeling procedure using frozen, unfixed tissue sections resulted in very dense labeling of the mammary gland. Binding sites for OXT were also found in various forebrain areas, including the hippocampus, especially the ventral subiculum and taenia tecta, central amygdala, posterior part of the anterior olfactory nucleus, claustrum, nucleus accumbens, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, and the posterior pituitary. The ependyma of the lateral ventricle and/or the chorioid plexus near the lateral septum was labeled as well. These data support the hypothesis that OXT plays a role in a number of centrally regulated processes.

Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets.

The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.

The bioavailability and metabolism of trilostane in normal subjects, a comparative study using high pressure liquid chromatographic and quantitative cytochemical assays

High pressure liquid chromatographic (HPLC) analysis of plasma taken over 8 h from ten normal male subjects medicated with 120mg of trilostane revealed that the drug is rapidly metabolised into at least one metabolite, 17-keto trilostane. Both compounds were detected in the blood stream at concentrations > 2 × 10 −7 M within an hour and were cleared from the blood by 6–8 h. Approximately 3 times the concentration of metabolite was detected compared to the parent compound in most samples analysed. There were large subject to subject variations in the handling of drug. Standard curves of pure 17-keto trilostane and trilostane were parallel as assessed by cytochemical bioassay. This assay is based upon the inhibition of 3β -hydroxysteroid dehydrogenase activity in unfixed tissue sections of the dioestrous rat ovary. The relative potency of the metabolite compared to trilostane was 1.71 (95% confidence 1.5–2.0) over the dose range 0.15–1.5 μM. Thus, the metabolite may be the major active agent when trilostane is administered for clinical purposes. In a further 4 volunteers, who also received 120mg trilostane and were sampled over an 8 h period, plasma was analysed independently by HPLC and cytochemical assays. In the majority of cases the bioactivity recorded (relative to a trilostane standard curve) was substantially higher than the molar sum of circulating trilostane and 17-keto-trilostane (as assessed by HPLC). However, if the relative potency of 17-keto-trilostane is taken into consideration, correlation between the two assays was excellent ( r = 0.947, n = 18, P < 0.001). This also suggests that no further active metabolites were present in the plasma samples. The drug profiles seen in the second study were essentially the same as described for the first 10 volunteers. The combination of a bioassay, which detects trilostane-like bioactivity, and HPLC, which reveals the type of metabolism, should aid our understanding of the clinical value of this potentially important drug.

Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

A cytochemical section bioassay for plasma trilostane: an orally active inhibitor of 3 beta-hydroxysteroid dehydrogenase activity.

Trilostane is a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase activity in vitro and an orally active inhibitor of steroidogenesis in vivo. A cytochemical section bioassay for this drug (4 alpha, 5-epoxy-17 beta-hydroxy-3-oxo-5 alpha-androstane-2 alpha-carbonitrile) in human plasma has been developed. The assay is based upon the inhibition of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the corpus luteum of unfixed tissue sections (6 micron) of the mature dioestrous rat ovary. Trilostane is extracted from plasma by a simple chloroform procedure. The standard curve (0.15-2.5 mumol/l pure trilostane) exhibits a four-fold change in 3 beta-HSD activity and is constructed in the presence of extract from control plasma pools. The detection limit of the plasma assay is 2 mumol/l trilostane after allowing for dilution effects and recovery losses. This level gives a significantly different response from the zero quality control (P less than 0.05) and has an inter-assay coefficient of variation of less than 15%. No false positives have been recorded to date and the majority of samples from subjects receiving therapeutic doses of the drug have levels of greater than 2 mumol/l. Intra- and inter-assay coefficients of variation using both plasma quality controls and unknown samples are 4% (n = 15) and 13.9% (n = 27) respectively. Recovery is 80 +/- 9% (n = 13) and 79 +/- 10% (n = 10) for plasma quality controls containing 12 and 4.8 mumol/l trilostane respectively. Dilutions (up to 1:5) of plasma extracts are parallel to the standard curve and the assay throughput is approximately 16 samples/week technician.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and localization of vasoactive intestinal peptide receptors in the rat lung.

Experiments were performed to characterize vasoactive intestinal peptide (VIP) receptors in rat lung and to study their localization by means of light and electron microscope autoradiography. [125I]Iodo-VIP binding to lung homogenate was inhibited by synthetic VIP, [Val5]secretin, secretin, and 7-27 secretin with half-maximal inhibition (ID50) values of 1.62, 13, 121 nmol/liter and up to 0.1 mmol/liter, respectively. The relative potency of [Val5]secretin and 7-27 secretin to displace the tracer indicated that [125I]iodo-VIP binds to specific VIP receptors. Kinetic studies showed two sites of binding, a site of high affinity with equilibrium dissociation constant (KD) values of 0.32 +/- 0.09 nM and Bmax of 88.7 +/- 28.6 fmol/mg protein and a lower affinity binding site with a KD value of 23.0 +/- 2.9 mM and Bmax of 584 +/- 136.9 fmol/mg protein. The localization of VIP receptors was performed by radioautography both in vivo after iv injection of [125I]iodo-VIP and in vitro by the direct application of the radiolabeled peptide to cryostat sections of unfixed lung tissue. After iv injection of label, silver grains were detected only over alveoli. Electron microscopy indicated that radioactivity was mostly associated with alveolar capillaries. In vitro localization studies showed specific binding not only to alveoli but also to bronchi. These studies suggest that VIP can act at different sites and that the peptide could gain access to alveoli via the general circulation.

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland.

A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37 degrees C in reaction medium containing 1.4 mM DAB (in 0.1 M Tris-HCl) and 0.15 mM hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10 mM. Stimulation of peroxidase in the thyroid was observed in vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.

20α-hydroxysteroid dehydrogenase activity in the rat corpus luteum; A quantitative cytochemical study

A quantitative cytochemical method for the demonstration of 20α-hydroxysteroid dehydrogenase activity (20α-HSD) in the regressing corpora lutea of the adult rat ovary is described. The method employs unfixed tissue sections and relies upon the oxidation of 20α-hydroxy-4-pregnen-3-one (20α-OH-P) with nitro blue tetrazolium as the hydrogen acceptor. The enzyme was dependent upon NADP + for its activity and was inactive when 20β-hydroxy-4-pregnen-3-one (20β-OH-P) was used as a substrate. The apparent K m values for 20α-OH-P and NADP + were 3 × 10 −4M and 2.5 × 10 −5M respectively. Inhibition of 20α-HSD activity by steroids was demonstrable at pH 8. Androstenedione was by far the most potent inhibitor, followed by progesterone (the product of the enzyme activity) 17α-hydroxyprogesterone. Compound S and 20β-OH-P. At pH 6.8, a pH more favourable to the progesterone → 20α-OH-P reaction, only progesterone and 17α-hydroxyprogesterone were inhibitory. Testosterone was without demonstrable effect at either pH.

Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality

A general technique is described for using slide-mounted unfixed tissue sections to characterize and visualize drug and neurotransmitter receptors in brain or other tissues. The preparation of material, from fresh frozen, unfixed brain to dried sections securely attached to slides, is described in detail. The tissue can be kept intact during incubation at varying temperatures in solutions containing radiolabeled ligand, ions, buffers, and allosteric effectors. Strategies are described for determining optimal stereospecific binding with highest signal-to-noise ratios and for determining that a "meaningful" receptor is being studied. Dry formaldehyde fixation by vapors from heated paraformaldehyde preserves the tissue quality and traps the ligand near its site on the receptor, permitting subsequent histological processing through alcohols, solvents, and aqueous media, including liquid nuclear track emulsion. Visualization of [3H]naloxone- or [3H]enkephalin-labeled opiate receptor distributions in rat and human brains is achieved by tritium-sensitive film or by classical "wet" emulsion autoradiography. The advantages of the film include its ease of use and the ability to quantify receptor density by densitometry which can be computer-assisted. The advantage of the emulsion is the greater resolution and the concomitant appearance of morphology in cell-stained sections. Examples of correlations of opiate receptor distributions which underlying cytoarchitecture illustrate the potential for receptor localization studies.

Cyclic Nucleotide Immunohistochemistry

It has been reported that cyclic nucleotide immunohistochemistry fails to localize all pools of cyclic nucleotides and to detect the changes in cyclic nucleotide concentration when applied to unfixed brain tissue sections. Losses of cyclic nucleotides during antibody incubations have been demonstrated, but it has not been determined whether such losses are due entirely to extraction or if phosphodiesterase activity contributes to the loss. We also found that cyclic nucleotides are extracted during the incubations required by the procedure but report that the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) reduces cyclic nucleotide losses.The application of the unlabelled antibody-enzyme technique and the inclusion of IBMX in incubation solutions permitted the detection of cyclic GMP in all layers of rat cerebellar cortex. The administration of ethanol (4g/kg, per os) ubiquitously reduced cyclic GMP staining.

Effects of dietary lipid and phenobarbitone on the distribution and concentration of cytochrome P-450 in the liver studied by quantitative cytochemistry

Cytochrome P-450 is an important component of the NADPH-dependent electron-transport system located in the endoplasmic reticulum of liver parenchymal cells and is the terminal oxidase involved in the oxidative metabolism of drugs, anaesthetics, carcinogens, steroid hormones, insecticides, environmental pollutants and fatty acids. In the reduced form this haemoprotein binds carbon monoxide to produce a characteristic Soret absorption band at 450 nm and is therefore routinely assayed in the microsomal fraction by measuring the difference spectrum of reduced cytochrome P-450 in the presence and absence of Carbon monoxide [ 11. It is well established that phenobarbitone administration increases the concentration of cytochrome P-450 in liver microsomes [2,3], and that variations in the content and composition of the dietary lipid can also markedly affect the concentration of cytochrome Pd.50 in liver microsomes [4,5]. Quantitative cytochemical measurements of the concentration of cytochrome P-450 in small groups of cells located in unfixed tissue sections using a Zeiss Universal Microspectrophotometer (type I) have shown that cytochrome P-450 is not evenly distributed in rat liver and is induced selectively in the centrilobular hepatocytes by phenobarbitone [6,7]. The effects of changes in dietary li$d composition on the distribution of cytochrome P-450 within the rat liver lobule have not been investigated.

Distribution of actively absorbed diffusible sugars in the jejunal epithelium of the rat.

Electron-microscopy autoradiography, using freeze-dried frozen sections of unfixed tissue, was used to study the distribution of actively transported materials in the jejunal epithelium of the rat in vitro. After a few minutes incubation, the grain density over the organelle-packed interiors of the apical cytoplasm of the columnar absorptive cells was significantly greater than that over the structureless peripheral cytoplasm. This difference in the relative specific activities of the 2 subcellular compartments increased during accumulation of labelled galactose, and decreased as preloaded galactose was washed out of the epithelium. A similar compartmentation was observed in vascularly perfused intestines exposed to labelled galactose from either the mucosal or the serosal sides. These observations suggest the presence of an intracellular mechanism controlling the location and concentration of transported substrates during intestinal absorption.

5' nucleotidase activity in the human synovial lining in rheumatoid arthritis.

5'-Nucleotidase (EC 3.1.3.5), a plasma membrane-bound enzyme, has been assayed in unfixed tissue sections of human synovium, activity being measured by scanning and integrating microdensitometry. Activity was markedly increased in the lining cells of the rheumatoid synovial membranes.

A quantitative study of N-acetyl-beta-glucosaminidase activity in unfixed tissue sections of the guinea-pig thyroid gland.

A post-coupling procedure for the quantitative measurement of N-acetyl-beta-glucosaminidase activity in unfixed tissue sections of guinea-pig thyroid is described. The method depends on the cleaving of a naphthol AS-BI substrate and the insoluble reaction product is post-coupled with Fast Garnet GBC salt (in acetate buffer, pH 6.2) at 4 degrees C. Even though this enzyme is localized in the lysosomes, an inert colloid stabiliser, polyvinyl alcohol (G18/140) is included in the reaction medium to allow the use of the optimal substrate concentration (0.5 mg/ml) whilst employing a low concentration (5%) of ethylene glycol monomethyl ether. The high molecular weight (90 000) grade of polyvinyl alcohol used did not stabilize the lysosomal membrane, although a lower molecular weight (15 000) grade of polyvinyl alcohol (G04/140) may do. The enzyme activity was not affected by the metal ions Ca2+ and Zn2+ and was totally abolished by the specific inhibitor 2-acetamido-2-deoxy-D-gluconolactone.

Formaldehyde-fluorescamine-induced fluorescence as a property of carcinoma cells.

Fluorescamine is a sensitive cytochemical probe for primary amino groups and produces an intense general fluorescence in unfixed tissue sections reflecting the ubiquitous occurrence of such groups. Following treatment with formaldehyde, most primary amino groups react to form derivatives unable to yield fluorescence with fluorescamine. Certain cell systems, however, contain amino groups which do not react with formaldehyde but display strong reactivity with fluorescamine. In formaldehyde- and fluorescamine-treated specimens such cell systems display an intense fluorescence, whereas the majority of tissue constituents are non-fluorescent. Fluorescent cell systems include certain protein- and peptide-secreting cells and a large number of different types of carcinoma cells. In some cases it appears that neoplastic transformation is necessary before the cells display formaldehyde-fluorescamine-induced fluorescence. Available data indicate that the reactive substance(s) are peptide in nature and that the production of such substance(s) may be a general property of carcinoma cells.

A quantitative cytochemical method for the demonstration of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary.

A method for the quantitative measurement of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD+ or NADP+ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP+ dependence is not abolished by inhibiting nonspecific alkaline phomonoesterase. The activity of delta5,3beta-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10(-4) M.

A quantitative study of the effects of different grades of polyvinyl alcohol on the activities of certain enzymes in unfixed tissue sections.

Different grades of the colloid stabilizer, polyvinyl alcohol, used for protecting unfixed cryostat sections during cytochemical reactions, may have different effects on enzymatic activity. The influence of three grades of polyvinyl alcohol on the activities of "soluble", membrane-bound and membrane-enclosed enzymes has been investigated in unfixed sections; the activities were measured microdensitometrically. The largest molecular weight polyvinyl alcohol (G18/140, mol. wt. about 90 000) did not retain glucose-6-phosphate dehydrogenase activity in sections of rat liver even when used at the maximum convenient concentration (12%); G04/140 and M05/140 (molecular weights of 15 000 and 25 000 respectively) retained this soluble enzyme if used at concentrations of 30 and 20% respectively. At these concentrations, lactate dehydrogenase activity was apparently decreased when G04/140 and M05/140 were used; this diminished activity has been shown to be due to the need to establish optimal concentrations of reactants for each grade of polyvinyl alcohol and for each reaction. When optimal concentrations of reactants were used, the activities of this enzyme in the presence of each grade of polyvinyl alcohol were identical. The presence of any type of polyvinyl alcohol did not influence the activities of mitochondrial succinate dehydrogenase or of the smooth endoplasmic reticulum enzyme, delta5,3beta-hydroxysteroid dehydrogenase. However, the presence of polyvinyl alcohol improved the state of the section.

The direct measurement of cytochrome P450 in unfixed tissue sections.

A method for the measurement of cytochrome P-450 in unfixed cryostat sections is described. The sections are incubated for 10 minutes at room temperature in a buffered solution containing polyvinyl alcohol and sodium dithionite. Two incubations are performed on serial sections, one in nitrogen and the other in carbon monoxide. Readings are taken on a Vickers M85 microdensitometer fitted with a high sensitivity photomultiplier amplifier system, the measurements being made on corresponding fields in the serial sections. Subtraction of the nitrogen values from the carbon monoxide values, after allowing for an absorption shift, gives the absolute spectrum of cytochrome P450. The subtraction corrects for the tissue content of other haem-containing proteins. The cytochrome P450 spectrum shows a sharp maximum at 450 nm, and two other minor components absorbing at 444 nm and 458 nm. The content of cytochrome P450 in animals fed with phenobarbitone was 2.4 times greater than in control animals.

Histochemical activation of rat-liver lysosomes by dimethyl sulfoxide.

The Gomori reaction for acid phosphatase was enhanced by inclusion of dimethyl sulfoxide (DMSO) in reaction media for incubation of cryostat sections of unfixed rat-liver tissue. The reaction was also detectably increased by DMSO in formol-calcium fixed tissue. Enhancment of the Gomori reaction for acid phosphatase by DMSO was reversible.