Studies in fluorescence histochemistry

Summary Pretreatment of sections of fixed tissue with selective blocking reagents for indigenous thiol groups did not, it was found, impair the fluorescence subsequently obtainable with the acetic anhydridesalicylhydrazide-zinc technique (Stoward and Burns, 1967) for localizing C-terminal carboxyl groups of proteins. This suggests that thiol and S-acetyl groups do not participate in the complex reactions involved in the method. In further support of this suggestion it was found that artificially introduced thiol groups also do not take part.Sites containing either, glycogen or neutral periodate-reactive mucosubstances did not fluoresce in sections subjected to the technique. This indicates that O-acetyl groups, although probably formed, are not involved in the reactions giving rise to the fluorescence reaction in proteins.     

Studies in fluorescence histochemistry

Summary Tissue proteins believed to contain a relatively high concentration of C-terminal carboxyl groups emit an intense blue fluorescence after being treated with first a hot mixture of acetic anhydride and pyridine, second salicylhydrazide and last zinc acetate. Characteristically they do not fluoresce when the zinc treatment is omitted. Muscular tissues emit the strongest fluorescence, but normally neither mucosubstances nor loose connective tissues fluoresce at all.These and other results are consistent with Barrnett and Seligman's view that acetic anhydride in the presence of hot pyridine transforms the C-terminal carboxyl groups of proteins into methyl ketones. They do not support Karnovsky's more recent theory that hot acetic anhydride more or less exclusively converts side-chain carboxyl groups of proteins into mixed acid anhydrides instead.     

Vacuumless freezing-drying: Its application in catecholamine histochemistry

Summary Details of a simple technique to prepare tissues for the catecholamine reaction are described. Fresh cryostat sections are dried above phosphorous pentoxyde at –25⁰C for 15 hours. Ice evaporates from the sections under normal atmospheric pressure at this temperature. Frozen-dried cryostat sections are treated with formaldehyde gas like other frozendried specimens and observed under the fluorescence microscope immediately. The patterns obtained are at least comparable with those obtained by the original Falck procedure.     

Microfluorimetric quantification of catecholamine fluorescence in rat sympathetic ganglia

Summary The formaldehyde-induced fluorescence (FIF) technique was used to generate catecholamine fluorphores in the perikarya of the sympathetic neurons in the superior cervical ganglion of adult rats. During microfluorimetric quantification, the photodecomposition was eliminated by a rapid measuring procedure with a small excitation field and by using only visible light between the measurements.The catecholamine fluorescence, induced in protein microdroplets with increasing noradrenaline concentrations, was linear up to 2×10^–2 M which exceeds the noradrenaline content of even the most intensively fluorescent neurons. Thus, the differences in fluorescence intensities directly reflect the physiological state of each neuron with respect to their catecholamine content. The mean histograms reveal the changes which can only occur in certain neurons, and which can disappear if the mean only is assessed. The microfluorimetric method was sensitive enough to detect even minute changes induced by reserpine treatment in the catecholamine content of the sympathetic ganglion cells.     

A new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.     

Formaldehyde induced catecholamine fluorescence in the mouse inferior laryngeal paraganglion

Summary The presence of high concentrations of catecholamines is shown in the mouse's inferior laryngeal paraganglion by means of fluorescence histochemistry. In mice, the entire organ is composed of 20 to 25 small, intensely fluorescent cells of oval shape (about 15 m in diameter). The paraganglion is well provided with capillaries. The identification of catecholamines in the inferior laryngeal paraganglion, originally described as nonchromaffin (parasympathetic) paraganglion, presents additional evidence that all paraganglia store biogenic amines, are related to the sympathetic nervous system, and belong to the APUD cell series.Supported by the Deutsche Forschungsgemeinschaft, Project No: Bo 525/1     

Dopamine in a molluscan nervous system: Synthesis and fluorescence histochemistry

The nervous system of the pond snail, Helisoma trivolvis, was investigated for its ability to synthesize and accumulate ³H-catecholamines from ³H-tyrosine. ³H-Dopamine, but not ³H-norepinephrine, was synthesized by several ganglia. The highest accumulations were found in the cerebral, pedal, and buccal ganglia. The Falck-Hillarp and glyoxylic acid fluorescence histochemical techniques were applied to the buccal ganglia to visualize dopamine-containing cells. Fluorescing cells were found on both dorsal and ventral sides of the ganglion. Peripheral nerves of the buccal ganglia also displayed catecholamine fluorescence and accumulated ³H-dopamine. However, no ³H-dopamine synthesis occurred in the cerebral-buccal connectives, which connect the buccal ganglia with the rest of the central nervous system. Therefore, we conclude that there is a dopaminergic system intrinsic to the buccal ganglia and their peripheral targets.     

Demonstration of cathepsin B in the rat kidney using fluorescence histochemistry

The procedure of mounting freeze-dried sections with celloidin was adapted for the fluorescent-histochemical demonstration of cathepsin B in the rat kidney. A good localization of reaction products was shown in freeze-dried, 5-micron sections which had been mounted free floating with 1.5% celloidin solution on albuminized slides. Using this procedure, the reaction products were localized in the lysosomes, particularly those of the convoluted proximal tubule.     

The value of the fluorescence histochemistry of biogenic amines in neurotoxicology

Conclusions Acid- and aldehyde-induced fluorescence offers a highly sensitive and specific instrument for the histochemical demonstration of biogenic amines. This technique can be used to advantage for the selective identification of those neuronal structures that contain biogenic amines, namely the peripheral postganglionic sympathetic neurones and the central aminergic neuronal systems.Structural changes of impaired aminergic neurones can be ascertained from their fluorescence microscope image and correlated with light and electron microscopical observations, so that selective neurotoxic changes, such as sympathetic denervation of organs, can be detected and the reversibility of these changes tested.The degree of functional and structural changes occurring in the above neuronal systems can be easily quantified by means of microfluorimetry.The histochemical approach is restricted by the necessity of using fresh and specially fixed tissues. The possibility of numerous pitfalls in the interpretation of histochemical reactions requires the simultaneous use of other optical methods, such as light and electron microscopy, or the testing of the uptake of exogeneous amines or their precursors, whenever the occurrence of neurotoxic effects is to be assessed.     

Combined fluorescence histochemistry and 3h-noradrenaline measurements of adrenergic nerves

Summary A method is described which combines the histochemical fluorescence technique of Falck and Hillarp with isotope measurements in the same pieces of tissue. Tissue pieces incubated in isotope solutions were treated for fluorescence microscopy and examined. They were then removed from the microscopical slides, and the radioactivity determined. It was shown that NA¹ content and estimated fluorescence intensity were well correlated. The procedure devised is of special value when isotope measurements are needed of structures which can be safely identified only in the fluorescence microscope, and it has been used for quantitative estimations of adrenergic innervation.Abbreviations used Na noradrenaline - cpm counts per minute - dpm desintegrations per minute     

Demonstration of catecholamine and resorcinolamine derivatives as formaldehyde-induced fluorescence in protein models

We assessed in protein droplet models the potential use of the formaldehyde condensation method for histochemical demonstration of a wide range of catecholamines and resorcinolamines. The experiments showed that all of the amines tested, except salbutamol and carbuterol, formed fluorophores, and that the fluorescence was specific [i.e., there was no fluorescence in the absence of formaldehyde, the fluorescence was quenched by water, and the fluorophores were subject to photodecomposition by the exciting (405-nm) light]. Peak wavelengths of the emission spectra were 480-485 nm for fluorophores of resorcinolamine derivatives. The fluorescence intensity of the catecholamines was greater than that of the resorcinolamines. Fluorophore formation was not hindered by substitution of t-butyl, phenylisoprophyl, or p-hydroxyphenylisopropyl on the amino-N in catecholamines (t-butylnorepinephrine, Cc24, Cc25, respectively) or resorcinolamines (terbutaline, Th1161, fenoterol, respectively), and fluorophores also formed for catecholamines with the amino-N in a ring structure (rimiterol) or with a long alkyl chain substituted on the amino-N (hexoprenaline). Our study showed that fluorescence microphotometry can be used to detect a range of drugs that are catecholamines or resorcinolamines, and hence it should be possible to use this technique to study the properties of dissipation of these amines in tissues.     

Detection of impervious tissue in tree bark with selective histochemistry and fluorescence microscopy

Use of conventional histochemical tests in conjunction with fluorescence microscopy has validated the concept of impervious tissue in the bark of trees. Application of phloroglucinol + HCl or toluidine blue O selectively quenched lignin autofluorescence and allowed visualization of intracellular suberin lamellae previously undetected. Fluorescence of intracellular lamellae was quenched with Sudan black B and enhanced with Sudan IV thus providing evidence for the suberized nature of a tissue heretofore regarded as nonsuberized.     

Magnesium and calcium ions enhance barotolerance of streptococci

Summary Magnesium and calcium ions were found to enhance barotolerance of Streptococcus faecalis ATCC strain 9790 growing in a complex, glucose-containing medium. Enhancement was indicated both by higher growth rates and yields at 408 atm, and also by an increase in the maximum pressure permitting growth from 550 to 700 atm. The optimum concentration of either ion was ca. 50 mM, and both ions appeared to be equipotent in affecting the same processes by chemically specific interactions. Sodium, potassium, strontium, manganous, chloride, bromide or sulfate ions were all ineffective or only marginally effective in enhancing barotolerance. Mg⁺⁺ and Ca⁺⁺ also enhanced growth of compressed, ribose-degrading cultures. Pressure increased the sensitivity of streptococcal growth to low pH, and there appeared to be two distinct effects of Mg⁺⁺ and Ca⁺⁺ on barotolerance. First, the rate of exponential growth was enhanced prior to the time at which culture acidity began to limit growth. Second, growth was possible in more acid conditions under pressure when the ions were present, and enhanced yields from compressed cultures were related to this partial reversal of the potentiating effect of high pressure on acid inhibition of growth.Barotolerance of Escherichia coli or Saccharomyces cerevisiae was not enhanced by these ions; while tolerance of two types of chain-forming cocci freshly isolated from a rotting mussel was enhanced.     

Pontamine sky blue: a counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry

The stain pontamine sky blue (PSB) has been shown to reduce background autofluorescence in catecholamine fluorescence and immunofluorescence histochemical preparations. Using PSB as a counterstain on whole-mount stretch preparations of human mesenteric blood vessels, a medium dense noradrenergic nerve plexus is clearly revealed, which previously had been only partially visible because of background autofluorescence. Image analysis of nerve densities in whole-mount stretch preparations of guinea-pig arteries containing noradrenergic, substance P-, and vasoactive intestinal polypeptide (VIP)-positive nerve plexuses shows that PSB staining does not alter the specific neuronal fluorescence and that it improves image definition.     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

The relationship between the intensity of combined formaldehyde-chloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

Summary The relationship between the intensity of combined formaldehydechloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.This study was supported by grant from J.K. Paasikivi Foundation.     

镁离子对黏液型铜绿假单胞菌生物膜形成过程的影响

目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物膜形成过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度,荧光探针FTTC-ConA染细菌胞外多糖（Extracellular Polymeric Substances,EPS）、荧光显微镜下观察各组多糖差别;SYTO9／PI染生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（Image Structor Analyzer,ISA）对各组生物膜结构参数进行定量分析。结果2d时,空白组和1mmol／L镁组的黏附细菌的荧光强度分别为1845．67±45．3和2254．78±42．45,t=-9．96,P〈0．05;0．1mmol／L的镁浓度下荧光强度也有增加,其余各时间组趋势与2d组相似;在荧光显微镜下观察可见随着镁浓度增加,EPS增多;激光共聚焦显微镜下可见随着镁浓度增加,生物膜活菌增加、菌落变密集;ISA软件分析结果示：空白组和1mmol／L镁组的6d生物膜厚度分别为（25．80±1．16）μm和（34．87±1．59）μm,t=-13．85,P〈0．05;区域孔率分别为0．96±0．05和0．90±0．04,t=2．48,P〈0．05;平均扩散距离分别为1．54±0．15和1．92±0．16,t=5．23,P〈0．05;结构熵分别为3．64±0．57和4．70±1．09,t=-2．6,P〈0．05,3d组生物膜也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附,影响随后生物膜的形成及结构。