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PubMed1975

A comparison of four quantitative cytochemical methods directed toward demonstration of DNA.

Populations of nuclei isolated from mouse brain tissue were stained by the following cytochemical methods considered stoichiometric for DNA: (1) the Feulgen reaction; (2) gallocyanin-chromalum after RNase; (3) pH 4.0 methylene blue after RNase; and (4) methyl green used in the presence of 2M magnesium chloride. Replicate preparations to be stained with gallocyanin-chromalum, methylene blue, and methyl green were acetylated prior to staining. All of these groups were examined by high-resolution scanning microspectrophotometry. The results indicated that of the methods examined, the Feulgen reaction, gallocyanin-chromalum used without prior acetylation, and methylene blue used with prior acetylation were the most useful in revealing differences attributable to variability in chromatin organization. The greatest variability in total extinction measurements was observed in acetylated, methylene blue-stained nuclei, while the least variability was observed in nuclei stained with methyl green in the presence of 2 M magnesium chloride. Acetylation produced different effects on dye-binding in different groups. It greatly increased binding in nuclei stained with methylene blue; it reduced binding in the methyl green-2 M magnesium chloride series.

Acetylation↗
PubMed1989

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation.

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.

Chemical Phenomena↗
PubMed1984

[Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine].

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoacridines↗
PubMed1977

A microfluorescent PAS method for the quantitative demonstration of cytoplasmic 1,2-glycols.

Fluorescent microspectrophotometry using dichroic mirror vertical epi-illumination of tissue sections stained with the PAS reaction (periodic acid and pararosaniline Schiff reagent) provides a measure of the relative concentration of 1:2 glycols within and between tissue sections. In PAS reacted sections of agarose gel, pararosaniline Schiff fluorescence increases linearly as the concentration of agarose increases (r=0.97, p less than 0.05). The concentration of glycogen within liver as measured by a phenol-based tissue assay is linearly correlated with pararosaniline Schiff fluorescence of formalin fixed liver sections (r-0.87, p less than 0.05). These relationships are unaffected by alcian blue or hematoxylin. Heretofore the amount of color reaction as measured by densitometry at the pararosaniline absorption peak was claimed to be an unreliable indicator of the amount of reactive glycol present in tissue. Our observations indicate that when the concentration of Schiff reagent exceeds an empiric limit relative to available polysaccharides, the Schiff reagent-tissue complex reflects light at the excitation wavelength instead of fluorescing the emission spectra. This can be circumvented by using dilute pararosaniline-Schiff reagent, shorteining the staining period, and lowering the temperature of the staining medium. While routine PAS staining reactions are followed by washing in running water to develop the red color seen with broad spectrum illumination, water development is unnecessary for the dye-tissue complex to fluoresce. The fluorescent emission peak and the maximum excitation peak of both developed and undeveloped pararosaniline-Schiff-reagent-tissue complexes are 645--50 nm and 540--45 nm, respectively. These spectral characteristics are not changed by binding to oxidation products of different glycoproteins or polysaccharides. Intense exposure to room light, but not 100 repetitive short (0.13 s) exposures, causes partial photodecomposition. Quantitative assessment of cytofluorescence requires definition of the optical system used to measure emission. In the microspectrophotometer employed in this study, dichroic mirrors reflect light with variable efficiency depending on wavelength from the light source to the stage, and variably block light reflected or emitted from the specimen, serving as crude barrier filters. These dichroic mirror characteristics are influenced by the exact nature of the optical coating on the surface of each individual mirror. Since the optical coating on the surface of each individual mirror. Since the optical coating of similar mirrors may vary, the properties of individual mirrors must be considered in the interpretation of spectral data and in determining the proper optical conditions for quantification of cytofluorescence.

Animals↗
PubMed1978

Cytospectrophotometric study of the kinetics of histochemical reaction of GABA-transaminase in cryostat sections of rat cerebellar cortex.

A modification of the histochemical method for demonstration of GABA-transaminase is proposed. Substrate and cofactor concentrations are chosen on the basis of kinetic investigation in cryostat sections of the rat cerebellar cortex. Enzymatic reactions were measured by quantitative microspectrophotometry. Michaelis constants for alpha-oxoglutarate in the Purkinje cell layer and granular layer (Km 1.7 x 10(-3) M) and white matter (Km 3.8 x 10(-3) M) are found. It is shown that alpha-oxoglutarate in concentrations higher than 5.2 x 10(-3) M (1 mg/ml) suppresses the reaction in sections by competitive inhibition. The advisability of addition of malonate, PMS and cyanide to the incubation medium is confirmed. It is suggested that there are some isoenzymes of GABA-transaminase with predominant localization either in neurons or glia.

Animals↗
PubMed1984

[Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect].

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining.

Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358 +/- 0.134 X 10(4) at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.

Animals↗
PubMed1989

Interspecific variation in the visual pigments of deep-sea fishes.

Visual pigments in the rods of 38 species of deep-sea fish were examined by microspectrophotometry. 33 species were found to have a single rhodopsin with a wavelength of maximum absorbance (lambda max) in the range 470-495 nm. Such visual pigments have absorbance maxima close to the wavelengths of maximum spectral transmission of oceanic water. 5 species, however, did not conform to this pattern and visual pigments were found with lambda max values ranging from 451 nm to 539 nm. In 4 of these species two visual pigments were found located in two types of rod. Some 2-pigment species which have unusual red sensitivity, also have red-emitting photophores. These species have both rhodopsin and porphyropsin pigments in their retinae, which was confirmed by HPLC, and the two pigments are apparently located in separate rods in the same retinal area. In deep-sea fishes the occurrence of 'unusual' visual pigments seems to be correlated with aspects of the species' depth ranges. In addition to ecological influences we present evidence, in the form of lambda max spectral clustering, that indicates the degree of molecular constraint imposed on the evolution of visual pigments in the deep-sea.

Animals↗
PubMed1988

The visual pigments of four deep-sea crustacean species.

The visual pigments of four mesopelagic crustacean species were studied at sea by means of microspectrophotometry. The absorbance maxima obtained for the visual pigments and their metarhodopsins, respectively, were: 493 nm and 481 nm (Systellaspis debilis), 485 nm and 480 nm (Acanthephyra curtirostris), 491 nm and 482 nm (A. smithi), and 495 nm and 487 nm (Sergestes tenuiremis). The spectral characteristics of the rhodopsins and metarhodopsins permit high photosensitivity and facilitate photoregeneration in a nearly monochromatic environment. Photic regeneration of rhodopsins from the deep-sea environment was demonstrated, and data were obtained which are consistent with the occurrence of dark regeneration. Specific optical density of the observed visual pigments was calculated for two species.

Animals↗
PubMed1985

Demonstration of chondroitin sulphate and glycoproteins in articular cartilage matrix using periodic acid-Schiff (PAS) method.

Staining of articular cartilage by the periodic acid-Schiff (PAS) method was measured using microspectrophotometry. Standard PAS technique with 2 h oxidation produced a distinct Schiff reaction in the cartilage sections. The staining increased with depth of the articular cartilage demonstrating distribution of the glycoproteins. The modified PAS method included a second, longer periodic acid treatment, which made the uronic acid of glycosaminoglycans PAS-positive. The modified PAS method proved to be highly specific for chondroitin sulphate, which was determined from the samples with gas chromatography. A statistically significant correlation between the Schiff reactivity and galactosamine content of the sections was observed. It is concluded that for articular cartilage standard and modified PAS methods are useful procedures for demonstrating local changes of glycoproteins and chondroitin sulphate, respectively.

Animals↗
PubMed1982

Sarcoplasmic Ca2+ transients during the contractile cycle of single barnacle muscle fibres: measurements with arsenazo III-injected fibres.

Single muscle fibres from Balanus nubilus were injected with the metallochromic Ca2+ indicator arsenazo III; multi-wavelength microspectrophotometry was used to monitor changes in the absorption spectrum of such fibres during isometric tension transients elicited by electrical stimulation. Differential absorbance changes, recorded in the 660-720 nm region of the spectrum, were characterized by rapid, nearly linear increases during constant current depolarizing pulses (25-200 ms); upon termination of electrical stimuli the increased differential absorbance described an exponential (k = 0.6-2.0 s-1) decay toward baseline levels. The maximal increases in these absorbance signals preceded the peaks of the tension transients by 300-400 ms at 15-17 degrees C. Calibration of these optical transients indicated that the initial velocity of the rising phase corresponded to a 0.03 microM ms-1 increase in sarcoplasmic free Ca2+. Appreciable tension was not generated until the apparent peak magnitude of the free Ca2+ transient exceeded 2 microM; a further three-fold increase (from 2 to 6 micrometers) in the maximal value of the free Ca2+ transient was accompanied by a 20-fold increase in the magnitude of the tension transient.

Animals↗
PubMed1977

The nuclear DNA content of lobular neoplasia of the mammary gland.

Twelve cases of lobular neoplasia and three cases of invasive lobular carcinoma of the breast were examined by Feulgen microspectrophotometry. Two different ranges of nuclear DNA values were observed. Eight cases of lobular neoplasia fell within the diploid range and the four remaining cases within the diploid to tetraploid range, while the three cases of invasive lobular carcinoma showed a DNA distribution between the diploid and tetraploid range. Cytophotometrically, lobular neoplasia with diploid DNA values can be considered to represent a precancerous condition, whereas lobular neoplasia with diploid to tetraploid values are regarded as a more advanced stage of an non infiltrating carcinoma still confined to the performed spaces.

Breast Neoplasms↗
PubMedDate not supplied

Regional asynchrony of segmental contraction may explain the "oxygen consumption paradox" in stunned myocardium.

Despite apparently depressed function, stunned myocardium maintains oxygen consumption and has the capacity to increase contractility with inotropic stimulation. We hypothesized that during stunning, O2 demand is maintained because regional segment work is performed, but is asynchronous with global left ventricular contraction, and that inotropic stimulation would restore regional work and synchrony. Thirteen open-chest anesthetized dogs were subjected to three left anterior descending (LAD) coronary artery occlusions (10 min) and reperfusions (15 min) to produce regional myocardial stunning. Segment shortening and force development were measured independently and simultaneously in the LAD (experimental) region and circumflex (control) regions. Regional myocardial work was calculated as the integrated product of instantaneous force and shortening, during two periods: 1) over the entire cardiac cycle (Positive Work), and 2) limited to the systolic portion of the cardiac cycle (Systolic Work). Regional myocardial O2 consumption (MVO2) was calculated from regional blood flow (radiolabeled microspheres) and O2 saturation data (microspectrophotometry). Occlusion of the LAD produced a delay in onset of segment shortening in the ischemic region, but not in regional force development. A time delay of 67-81 ms persisted through the three stages of occlusions and reperfusions. Systolic regional work was depressed to a greater extent (924 +/- 182 to 149 +/- 118 g*mm*min-1) than total positive regional work (1437 +/- 337 to 857 +/- 174 g*mm*min-1). Regional subepicardial MVO2 in the stunned region was not different than in the control region (7.3 +/- 1.5 vs. 6.9 +/- 1.4 ml O2*min-1*100 g-1). Local infusion of isoproterenol reversed the delay in regional shortening from 73 +/- 7 to 21 +/- 8 ms, thereby augmenting systolic work (298%) more than positive work (60%), without a significant increase in MVO2 (7.3 +/- 1.5 to 10.5 +/- 3.2 ml O2*min-1*100 g-1). It is concluded that myocardial stunning decreases regional systolic work due to regional mechanical asynchrony, while MVO2 is used supported total positive work which was not significantly reduced. Isoproterenol restores regional work by restoring synchrony, without greatly affecting regional MVO2.

Animals↗
PubMedDate not supplied

The oxygen wasting effect of isoproterenol is altered by chemical denervation and cardiac hypertrophy.

We tested the hypothesis that isoproterenol would increase myocardial work and O2 consumption at reduced efficiency and that both left ventricular hypertrophy and chemical sympathectomy would lead to changes in this myocardial efficiency response. Left ventricular hypertrophy was produced by aortic valve plication in 23 puppies. Six months later, sympathetic denervation (6-hydroxydopamine) was produced in 12 hypertrophied and 10 non-hypertrophied dogs, 5 days prior to acute experiments. Ten non-hypertrophied and 11 hypertrophied animals were not denervated. Measurements were made before and during an isoproterenol infusion (0.5 microgram/kg/ min). Regional myocardial work was calculated as the integrated product of force (miniature transducer) and segment shortening (ultrasonic crystals). Regional O2 consumption was calculated from regional blood flow (microspheres) and regional O2 saturations (microspectrophotometry). In all groups, regional O2 consumption increased with isoproterenol (non-hypertrophied, non-sympathectomized 6.5 +/- 0.8 to 20.3 +/- 5 ml O2/min/100 g, non-hypertrophied, sympathectomized 5.0 +/- 0.7 to 10.0 +/- 1.5, hypertrophied, non-sympathectomized 9.8 +/- 1.3 to 16.2 +/- 2.2, hypertrophied, sympathectomized 6.1 +/- 0.5 to 13.3 +/- 1.6). Regional segment work also increased in all groups with isoproterenol stimulation (non-hypertrophied, non-sympathectomized 781 +/- 73 to 1197 +/- 61 g.mm/min, non-hypertrophied, sympathectomized 996 +/- 221 to 2118 +/- 412, hypertrophied, non-sympathectomized 1031 +/- 145 to 3262 +/- 753, hypertrophied, sympathectomized 721 +/- 116 to 1745 +/- 402). In the non-hypertrophied, non-sympathectomized group, efficiency (work/O2 consumption) was significantly decreased from 122 +/- 17 to 76 +/- 9 g.mm/ml O2/100 g demonstrating an "oxygen wasting" effect. In the hypertrophied, non-sympathectomized group, segment efficiency significantly increased from 94 +/- 19 to 250 +/- 63. In both sympathectomized groups, efficiency was not altered by isoproterenol. Thus the oxygen wasting effect of beta-adrenergic stimulation was reversed by left ventricular hypertrophy and blocked by sympathectomy.

Animals↗
PubMed1997

Negative metabolic effects of cyclic GMP are altered in renal hypertension induced cardiac hypertrophy.

We tested the hypothesis that increasing myocardial cyclic GMP levels would reduce myocardial O2 consumption and that renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy would change this relationship. Four groups of anesthetized open-chest New Zealand white rabbits (N = 26) were utilized. Either vehicle or 3-morpholinosydnonimine (SIN-1) (10(-4) M, a guanylate cyclase activator) was topically applied to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. Guanylate cyclase activity was measured by conversion of GTP to cyclic GMP. 1K1C rabbits had a greater heart weight-to-body weight ratio (3.29 +/- 0.15) than controls (2.63 +/- 0.19). Systolic blood pressure was higher in 1K1C rabbits than in controls. In control rabbits, cyclic GMP levels (pmoles/g) were higher in SIN-1-treated (EPI: 7.5 +/- 1.6; ENDO: 8.1 +/- 1.5) than in vehicle-treated animals (EPI: 5.4 +/- 0.4; ENDO: 5.6 +/- 0.6). This effect was enhanced in 1K1C rabbits, with cyclic GMP levels in the SIN-1-treated group (EPI: 11.9 +/- 1.3; ENDO: 13.0 +/- 1.5) almost double those observed in the vehicle-treated group (EPI: 6.3 +/- 0.8; ENDO: 7.7 +/- 1.1). There were no significant differences in basal or maximally stimulated guanylate cyclase activity between controls and 1K1C rabbits. Myocardial O2 consumption (ml O2/min/100 g) was significantly less in the EPI region of control animals treated with SIN-1 (7.2 +/- 1.2) than in the same region of controls treated with vehicle (9.1 +/- 2.0). Myocardial O2 consumption was also significantly less in SIN-1-than vehicle-treated 1K1C animals (SIN-1-treated: EPI: 6.9 +/- 0.8; ENDO: 6.2 +/- 0.7; vehicle-treated: EPI: 10.0 +/- 0.8; ENDO: 12.5 +/- 3.0). There was no significant difference in O2 consumption between control and 1K1C animals after treatment with SIN-1. Thus, there was a greater elevation in cyclic GMP in 1K1C rabbits, but this did not result in a corresponding greater depression in O2 consumption. This suggests that cyclic GMP plays a role in the control of myocardial metabolism, and that the sensitivity of myocardial O2 consumption to changes in cyclic GMP is reduced by renal hypertension-induced cardiac hypertrophy.

Animals↗
PubMed1984

Quantitative cytochemistry of beta-galactosidase in normal and enzyme deficient (gal) pollen of Brassica campestris: application of the indigogenic method.

The available cytochemical methods for localization of beta-galactosidase have been evaluated using pollen grains of Brassica campestris. beta-Galactosidase-deficient pollen (gal), served as a control. Azo dye methods involving naphthyl substrates showed high and nonspecific background staining to the exine. The indigogenic method, employing 5-bromo-4-chloro-3-indoxyl beta-D-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction product, while mutant pollen grains remained colourless. Final reaction product formation was blocked by D-galactono-1,4-lactone, thus demonstrating the specificity of the enzyme reaction. Using microspectrophotometry, the absorbance of the final reaction product was found to be a linear function of incubation time and section thickness in cryostat sections up to 8 micron thick and was only slightly reduced by glutaraldehyde prefixation. The validity of the indigogenic method for quantitative analysis was confirmed by using an enzyme-containing polyacrylamide gel model system and enzyme-coupled Sepharose 4B beads. Cellular sites of enzymic activity have been determined using plastic sections: final reaction product occurred in the intine wall layer and peripheral cytoplasm.

Galactosidases↗
PubMed1981

Practical applications of the microphotometric quantification of histoenzyme reactions.

There are many histochemical reactions which can be used for the accurate topographic detection of enzymes in tissues. There are also many reports of applications of these techniques in which the results have been evaluated subjectively. However, only a few of these methods have been the subject of quantification, using different methodologies, most of them based on microphotometry. A critical analysis of these procedures has generally shown that they are valid either relatively or at an absolute level. Nevertheless, the routine application of these methods is not as widespread as one might expect. In this paper, the methodological developments that have taken place in the histochemical assay of some enzymes by microspectrophotometry are reviewed briefly. The enzymes include acid phosphatase, non-specific esterase, succinate dehydrogenase and cytochrome oxidase. These methods have given consistent and useful results when applied to our experimental models of irradiated skin, and thus seem useful for various other applications. The reasons why there have been so few applications for quantitative histochemistry are also discussed together with possible ways of making its use more general.

Acid Phosphatase↗
PubMed1980

The effect of histochemical methylation on the phosphate groups of nucleic acids: interpretation of the absence of nuclear basophilia.

The effect of hot methylation (hydrochloric acid-methanol)) on nuclear stainability was investigated in order to determine whether the progressive loss of basophilia is due to methylation of the diester phosphate groups of nucleic acid. DNA spots on filter paper were unchanged in their stainability towards Toluidine Blue even after methylation for 4 days, while RNA, chondroitin sulphate and hyaluronic acid lost their affinity for this dye after 4 h methylation. In formalin-fixed sections, methylation for 4 h led to a loss of nuclear basophilia. There was no concomitant increase in nuclear relative to cytoplasmic stainability with Fast Green FCF at pH 9, as judged from the use of a comparison eyepiece, evaluation of colour transparencies or by microspectrophotometry. In contrast, extraction with trichloroacetic acid prior to or after methylation led to a much improved Fast Green staining of nuclei, comparable to the staining obtainable after treatment with trichloroacetic acid alone. In conclusion, there is no evidence that hot hydrochloric acid-methanol, as used in histochemistry, methylates the diester phosphate groups of nucleic acids. The loss of nuclear basophilia can be explained as a result of the excess positive charge on the chromatin following methylation of all the protein carboxyl groups. This effect is maximal after 3-4 h treatment with acid methanol at 60 degrees C. Further methylation leads to depolymerization and extraction of DNA. RNA is depolymerized in less than 4 h.

Animals↗