Lysosomal enzyme activities in different types of amniotic fluid cells measured by microchemical methods,combined with interference microscopy

Summary In primary amniotic fluid cultures, four distinct types of cells were characterized as epithelioid (E I and E II), fibroblast-like (F), and large cells. Small numbers (1–200) of freeze-dried cells were isolated from colonies of each cell type and analyzed for the activity of three lysosomal enzymes: -N-acetylglucosaminidase, -galactosidase, and -glucosidase.When expressed per cell, the activities for each of the enzymes were not significantly different among the small types of cells (EI, EII, and F). However, 5 to 10-fold higher enzyme activities were found in the large cells. The dry mass of individual large cells, as measured by microinterferometry, was also 5 to 10 times higher than that of the smaller cell types.When expressed per unit of dry mass, the enzyme activities tested, appeared to be independent of the type of amniotic fluid cell. The significance of this observation for the rapid prenatal diagnosis of metabolic diseases is discussed.     

Studies in lipid histochemistry

Summary Conditions for a selective extraction of nonpolar lipids from tissue sections with acetone were investigated using methods of lipid chromatography, histochemistry and quantitative determination of lipid phosphorus.Extraction of nonpolar lipids is selective when water (present either in acetone or in tissue sections) is completely excluded from the extraction procedure and the extraction is carried out at 0–4° C for 20–30 minutes. Under these conditions a negligible amount of polar lipids is extracted which cannot be demonstrated histochemically. A very small amount of nonpolar lipids remaining in sections cannot be demonstrated histochemically either.A method for the preparation of anhydrous acetone was recommended and an extraction procedure devised. This is to be applied in cases where nonpolar and polar lipids are to be distinguished and as an integral part of all types of phospholipid stains.When water is present during the extraction procedure (either in acetone or in tissue sections) significant extraction of all polar lipids occurs which is proportional to the content of water, to the length of extraction and to the temperature. Under these conditions the extraction of nonpolar lipids is somewhat slower.The significance of selective extraction with anhydrous acetone in histochemical analysis of lipids is discussed particularly with reference to lipids in atherosclerotic plaques.     

Effect of freeze-drying and gamma irradiation on biomechanical properties of bovine pericardium

Freeze-drying and gamma irradiation are the techniques widely use in tissue banking for preservation and sterilization of tissue grafts respectively. However, the effect of these techniques on biomechanical properties of bovine pericardium is poorly known. A total of 300 strips of bovine pericardium each measured 4 cm × 1 cm were used in this study to evaluate the effect of freeze-drying on biomechanical properties of fresh bovine pericardium and the effect of gamma irradiation on biomechanical properties of freeze-dried bovine pericardium. The strips were divided into three equal groups, which consist of 100 strips each group. The three groups were fresh bovine pericardium, freeze-dried bovine pericardium and irradiated freeze-dried bovine pericardium. The biomechanical properties of the pericardial strips were measured by a computer controlled instron tensiometer while the strips thickness was measured by Mitutoyo thickness gauge. The results of the study revealed that freeze-drying has no significant (p > 0.05) effect on the tensile strength, Youngs modulus (stiffness) and elongation rate of fresh bovine pericardium. Irradiation with 25 kGy gamma rays caused significant decreased in the tensile strength, Youngs modulus and elongation rate of the freeze-dried pericardium. However, gamma irradiation has no significant effect on the thickness of freeze-dried bovine pericardium, while freeze-drying caused significant decreased in the thickness of the fresh bovine pericardium. The outcome of this study demonstrated that freeze-drying has no significant effect on the biomechanical properties of fresh bovine pericardium, and gamma irradiation caused significant effect on the biomechanical properties of freeze-dried bovine pericardium.     

Preparation of histochemical sections for quantitative determination of acid phosphatase activity by means of laser microanalysis

Summary Based on former experiments dealing with a quantitative assay of acid phosphatase (acP) activity in histological sections by means of a laser microanalysis. The authors present results of investigations concerning optimal conditions needed for this purpose. The investigations were performed on rat liver sections. The realtion between incubation time and depth of acP reaction appearing in tissue blocks was investigated along with the dependence of photometric readings on thickness of histological sections and on incubation times. From results of experiments it appears that optimal conditions for quantitative determination of acP activity using the proposed laser method are provided by sections 30 to 50 in thickness which have been incubated in the substrate containing medium for 45 min. Sections thinner than 30 are not recommended for quantitative assay with this method because of low accuracy of readings caused by too low amounts of reaction product present there.     

Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)     

Modification of the Falck-Hillarp formaldehyde fluorescence method using the vibratome®: simple,rapid and sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue

Summary Two modifications of the original Falck-Hillarp formaldehyde fluorescence technique are presented, both based on a recently introduced instrument, the Vibratome^®, which permits cutting of unembedded tissue with a section thickness down to 10 ,.The first modification involves sectioning of unfixed tissue at a temperature below +5°C, subsequent air drying and reaction with formaldehyde vapours. In the second procedure formalin fixed tissue is cut and processed as described above. It is essential that both formalin fixation and cutting of the fixed tissue takes place at a low temperature to avoid diffusion of the catecholamines.The results show that with both techniques central CA neurons can be visualized with a high degree of sensitivity. Furthermore, since the sections are free from fractures—a common problem in freeze-dried tissues—the Vibratome^® technique represents a valuable tool for mapping studies. It may also be added that since many steps of the original procedure are omitted the present techniques are also more rapid and simple. It is pointed out that using the Vibratome^® procedure on formalin fixed tissue, it will be possible to combine e.g. cholinesterase staining or Fink-Heimer silver impregnation or immunofluorescent studies with the Falck-Hillarp technique on serial or even on the same sections.     

Interferometric analysis of intrasection and intersection thickness variability associated with cryostat microtomy

Summary Mach-Zehnder interferometric measurements were used to assess the extent of section thickness variability (inter- and intrasection) associated with cryostat microtomy of adrenal sections over a typical working range of 10–20 m. Sections were obtained using a Bright's Cambridge rocking type and a Damon rotary type cryostat microtome to allow comparative analyses. The effective thickness of tissue sections after being mounted onto slides by flash drying was reduced by 90% relative to microtome section thickness setting. A linear relationship between measured thickness and microtome setting was obtained with both instruments. Thickness variability between replicate sections over the range of microtome settings approximated 11% for the rocking microtome and 5% with the rotary microtome. Average intrasection variability was found to be 7% for rocking microtome sections and 4% for sections obtained with the rotary microtome. However, this variability is a negligible source of error in cytophotometric analyses, providing replicate sections are used and an adequate number of measurements are made on mask-delimited individual cells or tissue specimen areas.     

Some characteristic reactions catalysed by the ribonuclease activity in sections from fixed tissues

Summary 1. The ribonuclease activity in sections from freeze-dried Carnoy-fixed rat pancreas has been characterized through studies on the products of interaction with synthetic nucleoside derivatives, nucleotide esters, and of the polynucleotide synthetic properties.2. Homogenates of the sections in McIlvaine's buffer atph 7.0 degrade pyrimidine 2:3-phosphates to the corresponding 3-dihydrogen phosphate, and the purine derivatives at a much slower rate.3. Synthetic cytidylyl-cytidine, and benzyl- and ethyl-3-esters of pyrimidine nucleotides are degraded to nucleoside 3-phosphates. All the purine, and the 2-esters of pyrimidine nucleotides are not affected during 24 hours of incubation.4. Under influence of the homogenates, ribopolynucleotides will readily be synthesized from cytidine-2:3-phosphate and cytidine, the 3-P-5dinucleotide links being identified in cytidylyl-cytidine and cyclic dicytidylic acid.5. The conclusion is reached that the activity of the sections coincides with the DEase and CPase activity of crystalline pancreatic RNase. Some aspects of the possibilities for the histochemical localization of the activity are briefly discussed.With 6 Figures in the TextAided by grants from the Royal Physiographic Society of Lund, the Nora Sörensson Foundation, and the J. V. and Charlotta Lundgren Foundation.     

Autoradiographic model experiments with 67Ga and 99mTc

Summary In order to evaluate the feasibility to localize correctly ⁶⁷Ga citrate and ^99mTc pertechnetate in tissues, the resolution of these radioactive compounds were measured in a model system using four different autoradiographic techniques, wet as well as dry.Wet autoradiographic techniques gave an almost complete loss of ⁶⁷Ga. In deparaffinized sections of fixed and paraffin-embedded tissue the remaining ⁶⁷Ga, which was probably bound to proteins, gave a resolution of 2.6 . ^99mTc was either completely lost in wet techniques or the procedure could not be performed at all because of the very short half life of ^99mTc.The resolution of ⁶⁷Ga in a dry autoradiographic technique (according to Stumpf) was 6.9 and the resolution of ^99mTc 22.6 .The technique in which frozen sections are thawed on dry film and consequently dryed, gave slightly better resolutions than the dry technique (according to Stumpf) with ⁶⁷Ga as well as ^99mTc.It is concluded that for the histological localization of ⁶⁷Ga and ^99mTc a dry technique is required. However, the use of a wet technique can be considered, provided a loss of the radioisotope is acceptable and the procedure is controlled by a dry technique.     

Ultrastructural localization of potassium and calcium in an insect ommatidium as demonstrated by X-ray microanalysis

Summary Ultrastructural localization of potassium and calcium in the ommatidium of the house-cricketGryllus domeslicus L. was studied by X-ray microprobe analysis using samples prepared as thin sections (2 or 5 m) of freeze-dried and embedded tissue. Real resolution was limited by the size of ice crystals (Fig. 2) and estimated as about 1 m.Average values for potassium, calcium, sodium and phosphorus in different cells of the compound eye are given in Table 1.Striking non-uniformity in distribution of these elements over the cells and their compartments was found by probe scanning (Figs. 3, 4, 5). The highest potassium and calcium concentrations were measured in the pigmented zones of photoreceptors and pigment cells. The pigment granules are thought to be the ionic depots of the eye.Potassium and sodium are fully accessible to water in sections of embedded tissue, whereas all the calcium and half of the phosphorus are not.The functional significance of the non-uniformity discovered is briefly discussed.     

Splitting of naphthol AS-BI β-galactopyranoside by acid β-galactosidase

Summary -Galactosidase hydrolyses naphthol AS-BI -galactopyranoside in a variety of rat and mouse organs using freeze-dried cryostate sections, hexazonium-p-rosaniline for simultaneous coupling and long time incubation. In comparison with the indolyl and naphthyl derivate the splitting rate of the naphthol AS compound is far lower.     

Reaction rate studies of glucose-6-phosphate dehydrogenase in rat tracheal epithelium; the effect of section thickness

Summary The reaction velocity of glucose-6-phosphate dehydrogenase has been quantified by continuous monitoring on a Vickers microdensitometer of the reaction product as it formed in sections of different thickness of rat tracheal epithelium. Reaction velocity was directly proportional to section thickness when either tetranitro BT or neotetrazolium was used as the final acceptor; the rate was the same with each tetrazolium salt.However, the amount of formazan deposited in a given time was not proportional to section thickness. When tetranitro BT was employed the reaction became non-linear in the thicker sections due to the inability of the instrument to record beyond a certain absorbance value. Using neotetrazolium a lag phase, due to the failure to overcome the critical supersaturation level of the formazan, preceded the linear response. The duration of this phase decreased as section thickness increased.The implications of these findings on studies using conventional end point methods of measurement are discussed.     

Immunocytochemistry of gonadotropic cells and identification of cell types in ultrathin cryosections of the pituitary of the rainbow trout,Salmo gairdneri

Summary The most frequently occurring cell types in the pars distalis of the pituitary gland of the rainbow trout, (i) the lactotropic, (ii) the gonadotropic, and (iii) the somatotropic cells, were identified in cryosections. Their morphological characteristics were compared with those of Epon-embedded material. Cell location, cell form, position of the nucleus, arrangement of rough endoplasmic reticulum and sizes of secretory granules proved to be useful parameters for identification. The size distribution of secretory granules of corresponding cells in cryosections and Epon sections proved to be similar. Additionally, both the immunoferritin and the unlabeled antibody enzyme method were applied for the immunocytochemical labeling of gonadotropic hormone-producing cells in cryosections. Anti-salmon-GTH as well as anti-carp-GTH serum showed the presence of GTH in both the smaller and the larger granules of the classical GTH cells, but also produced a reaction in TSH cells. Labelling of TSH cells was absent when using anti--carp-GTH. Specificity of the reaction depended upon the degree of dilution of the anti-GTH serum. Results with dilutions of 14,000 and 18,000 in the unlabeled antibody enzyme method, and of 18,000 up to 132,000 in the immunoferritin technique were optimal. Acid phosphatase activity in the smaller granules was demonstrated by enzyme cytochemistry in Epon sections. The relationship of the presence of hormone in these granules is discussed. The high sensitivity of the immunocytochemical labeling procedure is discussed with respect to cryo-ultramicrotomy.     

On the localization,the fine structure,and the chemical composition of crystalline inclusions in theThesium humifusum haustoria

Summary TheThesium humifusum haustoria onMedicago marina roots, fixed in October–November, frequently contain in the cytoplasm of their cells inclusions consisting of sticks 0,3 m in thickness and 8 to 10 m in lenght, alone or associated in stacks of 3 or 6 units. These sticks consist of fibres, 10 nm in thickness, oriented in the same direction and separated from the others by a gap of 8 nm; these fibres seem to be composed of helically wound filaments and a less electron-dense matrix.The chemical composition of these inclusions was studied by enzymatic digestion in ultra thin sections; pronase digested the cytoplasmic paracrystals. This demonstrates that they are composed primarily of protein.The physiological significance of the inclusions is discussed: presumably the haustorium functions as a storage organe.     

Polychromatic staining of epoxy semithin sections: a new and simple method

A simple, rapid method is described for the polychromatic coloration of semithin sections, which is applicable to material routinely processed for transmission electron microscopy. Material fixed with a glutaraldehyde-paraformaldehyde mixture and postfixed in osmium tetroxide with or without potassium ferrocyanide and embedded in different types of resin (Durkupan-ACM, Spurr resin, Taab resin) can be used. Constant and homogenous results are obtained with this technique, the staining procedure being achieved at room temperature in no more than 10 min. Sections of 0.5–1 m in thickness are oxidised and bleached. After washing, sections are stained in two steps with carbol methylene blue/carbol gentian violet solution and pararosaniline solution. Using the method described in this paper, a polychromatic coloration of the different cells and tissues was obtained (epithelial cells in various shades of blue-violet, connective tissue and elastic laminae of blood vessels in pink or red, etc.). This procedure provides greater contrast between cytoplasm and nuclei, and among the different types of cells and tissues than is seen with toluidine blue, which is very useful for observation and photography of semithin sections. Polychromatic methods found in the literature are normally complex and require a lengthy staining time or cannot be applied on material routinely processed for transmission electron microscopy. Our method is simple, rapid and can be used on any type of material routinely processed for transmission electron microscopy and embedded in epoxy resins.     

Histochemical detection of α-d-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-α-d-glucoside

Summary 5-Br-4-Cl-3-Indoxyl--d-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of -d-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fichsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid -d-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane -d-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane -d-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid -d-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal -d-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.Supported by the German Research Foundation (Sfb 174) and the BMFT (Project CMT 35)     

Thickness measurements of skeletal muscle sections using the light microscope

Synopsis A new device is described for improving the accuracy of measuring the thickness of cryostat sections by the focusing technique in the light microscope. The necessity of such measurements is demonstrated by the great variation (range 2.55 m–11.93 m) in the thickness of serial cross-sections of frozen muscle biopsies from 12 healthy men. The final dehydration of the sections was found to reduce the thickness of fresh sections by 47%. However, dehydration caused the cross-sectional area to be reduced by only 2.8%.     

A pressure test method monitors biofilm coated particle fluidization

A fluidized bed denitrifying reactor was run to examine the vertical segregation of sand particles on the basis of different biofilm coverage, so far neglected when modelling fluidized beds. The segregation was found to be significant and it can be directly correlated with the vertical hydrostatic pressure profile in the bed. A procedure was developed for the rapid determination of biofilm thickness from hydrostatic pressure data using a recently published method based on the use of the novel criteria expansion coefficient and specific occupied volume. A key feature of the procedure is the particle content, which can be calculated from particle characteristics and is correlated in this study with the hydrostatic pressure gradient. The method was verified by directly measuring biofilm thickness as a function of the vertical position in the bed. This way biofilm thickness can be calculated from a readily measurable hydrostatic pressure profile with an error of 0.04–0.06 mm. This error is believed to be due to N₂ gas entrapment in the denitrifying biofilm and to the original inaccuracy of the determination of particle size and volume. The method is rather insensitive to the exact biofilm density when the usual high-density carrier material is used.     

Determination of correction factors of 3H-β-self-absorption for quantitative evaluation of grain number in autoradiographic studies

Summary Deparaffinized and Feulgen-stained sagittal sections of the mouse brain were studied interferometrically in order to measure optical path differences of euchromatin and heterochromatin of various cell types. Furthermore, the ratio eu-: heterochromatin of each cell type was determined. From these data mass densities of karyoplasm and, finally, correction factors of ³H--self-absorption were calculated for comparing grain numbers of different cell types in quantitative autoradiographic studies after application of tritium-labelled substances. Remarkable differences of correction factors up to a factor of 2.18 were found. Furthermore, the actual section thickness was determined interferometrically. A reduction to about 0.60 of the microtome setting was measured in two different areas of the brain. Using mass densities together with actual section thickness correction factors for a thickness of 1 m were calculated. This was done also for cell types outside the brain the data of which were taken from literature. Thus, differences in correction factors up to about a factor of 4 were found pointing out the importance of considering ³H--self-absorption in quantitative autoradiographic studies.Dedicated to the sixtieth birthday of Prof. Dr. Brigitte Maurer-Schultze, Würzburg.     

Change in XET activities,cell wall extensibility and hypocotyl elongation of soybean seedlings at low water potential

In dark-grown soybean (Glycine max [L.] Merr.) seedlings, exposing the roots to water-deficient vermiculite (_w=–0.36 MPa) inhibited hypocotyl (stem) elongation. The inhibition was associated with decreased extensibility of the cell walls in the elongation zone. A detailed spatial analysis showed xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity on the basis of unit cell wall dry weight was decreased in the elongation region after seedlings were transplanted to low _w. The decrease in XET activity was at least partially due to an accumulation of cell wall mass. Since cell number was only slightly altered, wall mass had increased per cell and probably led to increased wall thickness and decreased cell wall extensibility. Alternatively, an increase in cell wall mass may represent a mechanism for regulating enzyme activity in cell walls, XET in this case, and therefore cell wall extensibility. Hypocotyl elongation was partially recovered after seedlings were grown in low-_w vermiculate for about 80 h. The partial recovery of hypocotyl elongation was associated with a partial recovery of cell wall extensibility and an enhancement of XET activity in the hypocotyl elongation zone. Our results indicate XTH proteins may play an important role in regulating cell wall extensibility and thus cell elongation in soybean hypocotyls. Our results also showed an imperfect correlation of spatial elongation and XET activity along the hypocotyls. Other potential functions of XTH and their regulation in soybean hypocotyl growth are discussed.