Reduction In Fluorescence Intensity Research Articles

Spectroscopic Studies of Oligonucleotide Adducts and Base Sequence Preference of Adducts Formed by the Stereoisomers of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-Tetrahydrobenzo[a]pyrene

Abstract 5′-d(CCTATAGATATCC) has been reacted with BPDE and the adducts derived from binding of BPDE to the exocyclic amino group of deoxyguanosine (dG) were studied with spectroscopic methods. The major dG-adducts of (+)- and (-)-anti-BPDE and a minor adduct of (+)-syn-BPDE showed the characteristics of trans-adducts. The major products formed with (+)- and (-)-syn-BPDE exhibit cis-adduct characteristics. Annealing of BPDE-modified oligonucleotides to complementary or partially complementary strands results in reduced fluorescence intensity in several cases and in others the intensity is markedly increased. These differences demonstrate that the adduct microenvironment is strongly influenced by duplex formation and interactions between the adduct and the base localized opposite the adduct. The presence of a BPDE-adduct (trans- as well as cis-adducts) strongly reduces the thermal stability of the duplexes. With the modified oligonucleotide duplex 5′-d(CCTATAGATATCC)/ 5′-d(GGATATCTATAGG), the cis-adducts o...

Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wild-type enzymes. The results show that the luminescence of W310 is red-shifted and substantially quenched relative to that of W84. It displays an average subnanosecond fluorescence lifetime (tau F) and a very short, 50 microseconds, room-temperature phosphorescence (RTP) lifetime (tau P). The perturbation of W310 luminescence is believed to arise from a stacking interaction with Y283. In contrast, W84 exhibits a fluorescence lifetime tau F of several nanoseconds and a long-lived phosphorescence lifetime tau P, typical of buried, unperturbed TrP residues. NAD+ binding to the tetrameric enzyme causes a 55% reduction of W310 fluorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence. W84, which is located far from the nicotinamide moiety of NAD+, is much less affected by the binding of the coenzyme; the reduction in fluorescence intensity is 35%, and its phosphorescence intensity is unchanged. Another consequence of NAD+ binding is a significant decrease of the RTP lifetime tau P of W84, manifesting thereby a conformational change in the region of the coenzyme-binding domain. However, no change is observed in the RTP lifetime tau P of W310 located in the catalytic domain. These findings and those obtained at partial coenzyme saturation support the conclusions derived from high-resolution crystallographic structures [Skarzynski, T., & Wonacott, A. J., (1988) J. Mol. Biol. 203, 1097-1118] that the NAD(+)-induced conformational change is sequential and that subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding.

Detection of Complex Genetic Alterations in Human Glioblastoma Multiforme Using Comparative Genomic Hybridization

The aim of the present study was to detect complex genetic alterations in human glioblastoma multiforme (GBM) by comparative genomic in situ hybridization (CGH). Of the 24 GBM that were examined, increased fluorescence intensities indicating chromosomal polysomy of chromosome 7 and gene amplification at chromosome 7p were found in 42% of the tumors. In addition, signal enhancement of chromosome 19 was present in 29% and at 12q13-15 in 21% of the tumors. We also detected reduction of fluorescence intensities indicating gross deletions on chromosomes 10 (58%), 9p (46%), and 13 (29%). There was a close correlation of CGH results when compared with Southern analysis of the EGFR gene localized on chromosome 7 and loss of heterozygosity detection of chromosome 9 and 10 by microsatellite PCR. A close correlation was also observed between copy number changes of chromosome 7 and deletions of chromosome 10. Amplification of chromosome 12q and deletions of chromosomes 9p and 13 seemed to be complementary in the tumors investigated in the present study.

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, GM3(Neu5Ac), GM3(Neu5Gc) and Gm1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was GM3(Neu5Ac) followed by moderately expressed GM3(Neu5Gc) and GM1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-GM3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle.

Focal and regional variations in the composition of the glycocalyx of large vessel endothelium.

The glycocalyx of the endothelium of the systemic arteries and vena cava of the rabbit was visualised by in situ perfusion fixation with glutaraldehyde containing Alcian blue. The thickness of the layer ranged from 45 +/- 1 nm in the coronary artery to 81 +/- 2 nm in the carotid. The glycocalyx was 20 +/- 1.5 nm thicker on the downstream side of intercostal ostia than on the upstream side. Changes in the staining pattern with increasing concentrations of MgCl2 indicated that carboxyl groups made the major contribution to the surface charge, though sulphate groups were also present, particularly in the aortic arch and carotid artery. Segments of the thoracic aorta and carotid artery were also stained in vitro with fluorescence labelled wheat germ agglutinin, and fluorescence intensity in histological sections was quantified using a video microscope equipped with a microcomputer-based image analysis system. The fluorescence intensity in the carotid was 1.65 +/- 0.15 times that in the aorta. Pretreatment with neuraminidase reduced fluorescence intensity by 60 +/- 4% in the carotid and 53 +/- 2% on the upstream side of intercostal ostia, but only by 37 +/- 3% on the downstream side. Chondroitinase and heparanase both reduced binding and when used together their effect was additive, reducing fluorescence by 27 +/- 3, 51 +/- 4, and 32 +/- 3% at the three sites, respectively. Though the interpretation of the lectin binding experiments is complicated by a number of factors, these results support previous reports that sialyl groups are abundant in the endothelial glycocalyx. Glycosaminoglycans are also present, however, in significant amounts.

Characterization of Starch Granules in Maize Using Microspectrophotometry

AbstractStarch granule‐associated proteins and protein aggregates not associated with starch granules were visualized by staining with acid fuchsin. Based on fluorescence intensity, it was observed that non‐granule‐associated proteins could be removed from starch granule preparations by treatment with a modification of the Osborne‐Mendel fractionation method without affecting the apparent level of granule‐associated proteins. Moreover, fluorescence microscopy with direct spectrophotometric measurement of the proteins confirmed the presence of major subpopulations of granules differing in size and acid fuchsin‐induced fluorescence. Treatment of stained granules with 1.0% SDS dramatically reduced fluorescence intensity, indicating the removal of granule‐associated proteins.

Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.

To ascertain the ability of commercial and home-made anti-fading media to reduce the decrease of fluorescein isothiocyanate (FITC) fluorescence, we studied the bleaching characteristics of FITC-stained Reh 6 cells mounted in buffered glycerol and in anti-fading media. We measured the intensity of fluorescence over time with a confocal laser scanning microscope and a standard epifluorescence microscope coupled to an image analysis system. Most of the anti-fading media effectively retard fading but each has drawbacks. Better results were obtained with media containing p-phenylenediamine (solutions in buffered glycerol, Vectashield, Fluorstop). However, Mowiol, Slowfade, n-propyl gallate (20 g/liter) were also effective in retarding fading. Most of them, except Mowiol, reduced fluorescence intensity. We concluded that the choice of anti-fading medium would depend on the desired results: a slower decay of fluorescence despite an initial quenching of fluorescence or a lower retardant effect with no decrease in initial fluorescence intensity. Moreover, the combination of Mowiol with another anti-fading medium may be a useful compromise when a strong retardant effect is required without marked quenching of the initial fluorescence.

Fluorescent chemosensing of catechol and catecholamines in water

It has been known for almost 35 years that catechol complexes reversibly to boronic acids. We observe that 2-anthrylboronic acid complexes catechol in water with K d 330 μM and concomitant 20-fold reduction in fluorescence intensity, l-DOPA and dopamine behave similarly, suggesting a mechanism for the development of real-time sensing schemes.

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 microgram/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 microgram/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate-labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization–sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 νg/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 μg/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate–labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule. These results suggest that nitric oxide mediates the lipopolysaccharide-activated, Kupffer cell–induced inhibition of mitochondrial electron transport in hepatocytes. (HEPATOLOGY 1993;18:380–388).

α 2-Macroglobulin bait region integrity : Role in determining fast-form structure

To determine whether integrity of the bait region affects the structure of the remainder of human α 2-macroglobulin (α 2M), we have determined the separation between cysteine residues in a methylamine-reacted fast-form of α 2 M. From reduction in fluorescence intensity of covalently-bound donor fluorophore caused by proximity to an acceptor, a separation of 35 ± 8 Å was calculated, which is identical to a previously determined value for proteinase-treated fast-form α 2M. This indicates that although bait region cleavage is the physiological route to conformational change in α 2M, bait region integrity per se does not significantly affect the structure of fast-form α 2M.

Picosecond fluorescence decay in photolyzed lens protein alpha-crystallin.

Photolysis of calf lens protein alpha-crystallin in aqueous solutions has been monitored by observing changes in fluorescence decay following UV irradiation at 308 nm. The fluorescence decay was biexponential in dark controls and in photolyzed solutions. The recovered lifetime components in pH 7.4 phosphate buffer at 23 degrees C were 3.5 and 0.5 ns before irradiation and 2.7 and 0.5 ns after irradiation. As the UV dose increased, the relative weighting coefficient of the 2.7-ns decay component decreased, and that of the 0.5-ns component increased, resulting in an overall lifetime shortening. Similar results were obtained in 5 M guanidine hydrochloride solutions where lifetime components of 2.7 and 0.5 ns were observed. These observations were in contrast to the behavior of tryptophan monomer solutions which did not show any change in fluorescence decay kinetics upon UV photolysis but only a reduced fluorescence intensity. Steady-state fluorescence spectra and fluorescence quantum yields were also measured at 23 degrees C for unirradiated bovine alpha-crystallin and gave phi F = 0.11 +/- 0.01 in pH 7.4 buffer and phi F = 0.10 +/- 0.01 in 5 M guanidine hydrochloride solutions. The combined steady-state and fluorescence decay data were consistent with assignment of the long-lived fluorescence decay component in alpha-crystallin to emission from Trp-9, which is known to photolyze relatively rapidly. The short decay component was assigned to Trp-60, which photolyzed much more slowly. We thus provide an example of using steady-state photochemical data to assign fluorescence decay components in a multi-tryptophan protein.

Competitive Measurement of Rate Constants for Hydroxyl Radical Reactions Using Radiolytic Hydroxylation of Benzoate.

Hydroxyl radicals were generated from N2O-saturated phosphate-buffered saline solution (pH 7.5) by irradiation with a 3.7 TBq 137Cs source. The radicals attacked benzoate to form highly fluorescent products. The induction of fluorescence was tested in 0.002 to 2mM of benzoate solution; the fluorescence intensity was approximately constant at benzoate concentrations more than 0.1 mM during irradiation, and increased linearly with irradiation dose up to 53 Gy tested. The major primary products detected by high-performance liquid chromatography were three monohydroxybenzoates, 2-, 3- and 4-hydroxybenzoate (HOBZ). The G-values obtained from γ-irradiation for 60 min of a 0.2 mM benzoate solution were G(2-HOBZ)=0.97, G(3-HOBZ)=0.48 and G(4-HOBZ)=0.45. As the fluorescence of irradiated benzoate solution arose mostly from 2- and 3-HOBZ and the intensity of 2-HOBZ is 30 times that of 3-HOBZ, 98% of the fluorescence intensity induced was ascribed to 2-HOBZ. A hydroxyl radical scavenger competed with benzoate for hydroxyl radicals produced, and diminished the fluorescent products. A rate constant for the reaction of a scavenger with hydroxyl radical could be determined from the reduction in fluorescence intensity, using 0.2 mM benzoate and various concentrations of the scavenger. The fluorescence intensity was detectable down to 0.15 μM of 2-HOBZ produced. For various compounds, rate constants obtained in this way were similar to those measured by pulse-radiolysis.

Microtubule behavior in PC12 neurites: Variable results obtained with photobleach technology

We have examined the effects of various means of photobleaching on the recovery of fluorescence, movement, and morphology of the microtubules in the neurites of rhodamine-tubulin-injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low-power beam for an extended period, fluorescence in polymer recovers very little after 1 hour. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high-power laser, for 0.5-5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate-stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high-intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20-30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extend through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in PC12 neurites, and both are likely to be involved in neurite maintenance and growth.

A Molecular Fluorescence Spectrometer That Corrects for the Effects of Quenching

A fluorescence spectrometer has been constructed that simultaneously determines fluorescence intensity and excited-state lifetime. The ratio of intensity to lifetime is used to correct for the reduction in fluorescence intensity caused by collisional quenching. A frequency-domain technique is used to determine the excited-state lifetime in real-time. Computer-driven data acquisition provides instantaneous readout of the fluorescence intensity, the excited-state lifetime, and the ratio of these quantities. Correction for quenching by oxygen is demonstrated.

Analysis of various types of chronic granulomatous disease with the monoclonal antibody 7D5 directed against the small subunit of surface cytochrome b558

Four different types of chronic granulomatous disease (CGD) were analysed with the monoclonal antibody, 7D5, directed against the small 23 kD subunit of cytochrome b558 using a flow cytometric fluorescence analytical method. 7D5 immunofluorescent surface staining of granulocytes was absent in 12 patients with X-linked cytochrome b558 deficiency, in 2 patients with variant X-linked CGD with residual (X-forming activity and in 2 patients with autosomal recessive cytochrome b558 deficiency. The mothers of the patients with the X-linked form of CGD had 2 cell populations, one 7D5 negative or weakly positive and one 7D5 positive. The granulocytes of both parents of one patient with autosomal recessive cytochrome b558 deficiency had slightly reduced fluorescence intensity comparable to their reduced cytochrome b558 content. Three CGD patients with normal cytochrome b558 and their parents had granulocytes normally stained with antibody 7D5. 7D5 antibody enables rapid detection and classification of CGD patients with cytochrome b558 deficiency as well as rapid identification of heterozygous carriers.

Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion

The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with α-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31–49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.

Fluorescence banding in mitotic and meiotic chromosomes of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae)

Mitotic and meiotic chromosomes of the Mediterranean fruit fly, Ceratitis capitata, were studied using three counterstain-enhanced fluorescence staining methods. The tristaining technique allowed chromomycin A3 (CMA) and distamycin – diamidinophenylindole (DA–DAPI) fluorescence to be observed on the same chromosomes. DAPI–actinomycin D (DAPI–AMD) fluorescence was also carried out. These techniques were complemented with quinacrine staining and C-banding. The results were compared with earlier data on silver staining. The sex chromosomes, particularly the X chromosome, show great banding detail with extensive longitudinal differentiation in mitotic chromosomes. GC- and AT-specific fluorescence is not found in the expected reciprocal pattern at all sites. Comparison with C-banding and silver staining shows that intense fluorescence occurs in lightly C banded regions and silver bands correspond to fluorescent bands rather than nucleolar organizers. The combination of staining data suggests that much of the X chromosome has characteristics intermediate between heterochromatin and euchromatin. Meiotic X chromosomes show much less detail and reduced fluorescence intensity but can still be easily traced throughout meiosis and spermatogenesis.Key words: fluorescence banding, sex chromosomes, Mediterranean fruit fly, Ceratitis capitata.

Fluorescence properties of hog kidney aminoacylase I.

The state of the tryptophan residues of porcine kidney aminoacylase I (EC 3.5.1.14) was investigated by fluorescence spectroscopy and chemical modification. The pH-dependence of the fluorescence emission spectrum of the enzyme indicates that its native conformation prevails between pH 6 and 9.5. Within this range, the ionization of a residue with an apparent pKa of 7.1 quenches the enzyme fluorescence by about 15%. A similar reduction of fluorescence intensity accompanies the inactivation of aminoacylase I by treatment with N-bromosuccinimide in low excess. This suggests that in both cases a single tryptophyl residue out of eight residues per subunit is affected. Quenching by iodide revealed that, in the native conformation of the enzyme, 5-6 tryptophans per subunit are accessible, while 2-3 are buried within the protein. 8-Anilinonaphthalene-1-sulfonate (ANS) is tightly bound to aminoacylase I (1 mol/mol dimer, Kd less than 1 microM). ANS binding does not interfere with substrate turnover; the spectroscopic properties of the aminoacylase-ANS complex are consistent with bound ANS being excited by radiationless energy transfer (RET) from buried tryptophyl residues of the enzyme.

Bromobimanes--fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections.

Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient. In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons--not stainable by the classical methods--which can be identified as peptidergic ones by the bromobimane technique. A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.