Osmium Tetroxide-Potassium Ferrocyanide Intensification of a Diaminobenzidine Product Obtained by Photoconversion of a Fluorescent Label: a Study of Human Neutrophil Granules

A diaminobenzidine posttreatment employing osmium tetroxide and potassium fer-rocyanide was successfully used to intensify the diaminobenzidine stain formed by photoconversion of immunofluorescent labelling. Lactoferrin labelled granules became visible following the photoconversion process. Adequate diaminobenzidine staining was obtained after irradiating the cytospin preparations with ultraviolet light for 30-40 min. The diaminobenzidine stain had advantages over the fluorescent stain owing to its greater stability, greater density, and ability to be intensified. The enhancement procedure intensified both the color and density of the diaminobenzidine product. Consequently, shorter irradiation times could be used. Osmium tetrozide solutions of 3-4% increased the intensity with minimal background staining. Ultrastructural immunogold cytochemistry on ultrathin sections confirmed the existence of the lactoferrin labelled structures observed by light microscopy of cytospin preparations indicating that these were secondary grannies. The photomicroscopy procedures used in this study were simple to perform and could be applied to studies of other cellular antigens prior to using Immnnoultramicroscopy.     

Visualizing Endocytotic Pathways at Transmission Electron Microscopy via Diaminobenzidine Photo-Oxidation by a Fluorescent Cell-Membrane Dye

The endocytotic pathway involves a complex, dynamic and interacting system of intracellular compartments. PKH26 is a fluorescent dye specific for long-lasting cell membrane labelling which has been successfully used for investigating cell internalization processes, at either flow cytometry or fluorescence microscopy. In the present work, diaminobenzidine photo-oxidation was tested as a procedure to detect PKH26 dye at transmission electron microscopy. Our results demonstrated that DAB photo-oxidation is a suitable technique to specifically visualise this fluorescent dye at the ultrastructural level: the distribution of the granular dark reaction product perfectly matches the pattern of the fluorescence staining, and the electron density of the fine precipitates makes the signal evident and precisely detectable on the different subcellular compartments involved in the plasma membrane internalization routes.Key words: Endocytosis, PKH26 dye, diaminobenzidine photo-oxidation, transmission electron microscopy     

Novel application of tyramide signal amplification (TSA): ultrastructural visualization of double-labeled immunofluorescent axonal profiles.

Fluorescent immunocytochemistry (FICC) allows multiple labeling approaches when enzyme-based techniques are difficult to combine, such as in double-labeling experiments targeting small-caliber axonal segments. Nevertheless, the conversion of FICC to a product visible at the electron microscopic (EM) level requires labor-intensive procedures, thus justifying the development of more user-friendly conversion methods. This study was initiated to simplify the conversion of FICC to EM by employing the unique properties of tyramide signal amplification (TSA), which allowed the simultaneous targeting of a fluorescent tag and biotin label to the same antigenic site. Briefly, one of two antigenic sites typically co-localized in damaged axonal segments was visualized by the application of a fluorescent secondary antibody, with the other tagged via a biotinylated antibody. Next, an ABC kit was used, followed by the simultaneous application of fluorophore-tyramide and biotin-tyramide. After temporary mounting for fluorescent digital photomicroscopy, sections were incubated in ABC and reacted with diaminobenzidine before EM analysis. Double-labeling fluorescent immunocytochemistry with TSA clearly delineated damaged axonal segments. In addition, these same axonal segments yielded high-quality EM images with discrete electron-dense reaction products, thereby providing a simple and reproducible means for following fluorescent analysis with EM.     

A tetramethylbenzidine/tungstate reaction for horseradish peroxidase histochemistry

Tracing of neuroanatomical pathways commonly involves the histochemical demonstration of horseradish peroxidase, using the chromogen tetramethylbenzidine. A new modification of this reaction using ammonium paratungstate stabilizer retains high sensitivity while permitting the reaction to be performed at pH 6.0 in isotonic solutions. The reaction product resists solvents, allowing Nissl-stained sections to retain their peroxidase labeling. With subsequent stabilization by diaminobenzidine, the tissue is suitable for electron microscopic study and is compatible with post-embedding immunocytochemistry.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

Summary The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid, choline acetyltransferase or serotonin

In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gamma-amino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H2O2 (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H2O2 (brown reaction product in target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.     

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.     

Cloning of AFLP markers detected by fluorescence capillary electrophoresis

The available methods to isolate specific amplified fragment length polymorphism (AFLP) markers can be used only if markers are detected by radioactive labeling, silver staining, or ethidium bromide staining; these methods are useless if modern and automated genetic analyzers are used to detect AFLP markers by fluorescent labeling. We have developed a method that allows for isolation and cloning of specific AFLP markers obtained with a laser-induced fluorescence capillary electrophoresis system. This procedure has been tested on 5Arabidopsis thaliana polymorphic AFLP markers, and the nucleotide sequences obtained from these cloned markers were identified and located in theArabidopsis genome.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

An in vivo 5'-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%-20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy

We have previously shown that a fluorescent derivative of ceramide, N-(epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro-sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.     

A LIGHT AND ELECTRON MICROSCOPIC STUDY OF PEROXIDASE LOCALIZATION IN THE ONION ROOT TIP

Peroxidase activity in the onion root tip was localized by the diaminobenzidine procedure and studied at the light and electron microscope levels. Reaction product was observed in the cell wall, on the plasmalemma, in the Golgi apparatus cisternae and vesicles, in young and developing vacuoles, in the endoplasmic reticulum, and on both soluble and membrane-bound ribosomes. The reaction product at the various cellular and subcellular sites occurs in distinct tissue and developmental patterns.     

Ultrastructural localization of cholesterol by enzyme histochemistry

Summary The histochemical localization of cholesterol using oxidized diaminobenzidine as the final reaction product was studied at the electron microscopical level and compared with the digitonin method of cholesterol localization based on cholesterol digitonide as the final reaction product. Tissue chopper sections of fixed rat adrenal glands were incubated at 37° C in a medium consisting of 0.8 units/ml cholesterol oxidase, 1.4 units/ml cholesterol ester hydrolase, 50 units/ml horseradish peroxidase, 0.5 mg/ml diaminobenzidine, 0.1% v/v Triton X-100 (or Surfal) and an endogenous peroxidase inhibitor in 0.1m phosphate buffer, pH 7.0. An electron-dense osmiophilic reaction product was observed in many lipid droplets, intracellular vesicles and focally around mitochondria. Appropriate control experiments indicated that deposition of reaction product depended on the presence of cholesterol and the necessary enzymes. Comparison studies using digitonin confirmed the presence of cholesterol in the lipid droplets, but ultrastructural distortion limited the resolution of the more discrete deposits of cholesterol such as around mitochondria. The enzyme method permits finer resolution of these discrete deposits of cholesterol than the digitonin method because it does not cause distortion of cellular ultrastructure attributed to the formation of cholesterol digitonide. The enzyme method or a combination of enzyme and digitonin enables localization of free, esterified or total cholesterol.     

Simultaneous characterization of efferent and afferent connectivity, neuroactive substances, and morphology of neurons.

We present a method for establishing in a single experiment four characteristics of individual neurons: the efferent and afferent connectivity, the morphology, and the content of a particular neuroactive substance. The connectivity of the neurons is determined by retrograde fluorescent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-micron thick slices. Neurons containing retrogradely transported Fast Blue are intracellularly injected with the fluorescent dye Lucifer Yellow to fill their dendritic trees. The slices are then resectioned at 20-40 microns. One section through the soma of a Lucifer Yellow-filled neuron is selected for the detection of a neuroactive substance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filtering, it can be determined in the fluorescence microscope whether a Lucifer Yellow-containing cell body has also been labeled with TRITC, i.e., whether it is immunoreactive for this neuroactive substance. The adjacent sections are subjected to dual peroxidase immunocytochemistry with different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer Yellow (diaminobenzidine, brown reaction product) in the dendrites of the intracellular injected cells. The other sections are used for electron microscopic visualization of the transported PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow-injected, retrogradely Fast Blue-labeled cells (brown color) are studied by light microscopy. The electron microscope supplies ultrastructural data on the PHA-L-labeled axon terminals.     

A New Low Cost Wide-Field Illumination Method for Photooxidation of Intracellular Fluorescent Markers

Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our “photooxidizer” consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy.     

Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid,choline acetyltransferase or serotonin

Summary In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gammaamino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of primary antisera, the sections are incubated in a cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H₂O₂ (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H₂O₂ (brown reaction product in the target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver

Summary The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light-and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mm diaminobenzidine, 44 mm hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 m. Catalase in rat liver showed a K_m value of 2.0 mm for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mm. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.