A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.     

A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride

Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.     

Ultrastructural electron probe X-ray microanalytical reaction product identification of three different enzymes in the same mouse resident peritoneal macrophage

Summary Simultaneous cytochemical enzyme localization procedures for peroxidase (PO) plus acid phosphatase (AcP-ase) and/or aryl sulphatase (AS) have been investigated at the ultrastructural (EM) level. Electron probe X-ray microanalysis (EPMA) will identify and differentiate the reaction products.Dual reaction product localization of PO plus AcP-ase or alternatively PO plus AS have been obtained in the same mouse resident peritoneal macrophage. This has been acquired by first performing a PO-reaction followed by AcP-ase or followed by AS. In both cases PO-related reaction products (PO^DAB/Os or PO^DAB/Pt) were localized in nuclear envelope (NE) and rough endoplasmic reticulum (RER). Cells were identified by this reaction product as resident macrophages. Reaction products from the AcP-ase related cerium (AcP-ase^Ce), localized in lysosomes have been identified and differentiated from the PO-related osmium containing products. Similarly AS related barium (AS^Ba), localized in lysosomal structures and (R)ER was identified and differentiated.Triple reaction product localization of PO followed by AcP-ase plus AS could also be obtained. In this case, PO-related platinum containing reaction products (PO^DAB/Pt or PO^DAB/Os) in NE and RER has been identified and differentiated from the AcP-ase related lysosomal cerium (AcP-ase^Ce) and the AS related barium localized in lysosomal and (R)ER structures.Reversing the sequences in both dual cytochemical procedures: AcP-ase^Ce or AS^Ba followed by PO^DAB/Os (or PO^DAB/Pt) resulted in AcP-ase^Ce or AS^Ba activity related reaction products only. Reversing the sequence in the triple reaction procedures (AS^Ba followed by AcP-ase^Ce) resulted in the absence of the barium containing reaction products.By application of OsO₄ postfixation with aminotriazole (ATR) additives the detrimental effects upon the various precipitates have been confirmed.In LM studies, using rat intestine and non-metal identification reactions for two of the enzymes (pararosaniline for AcP-ase, DAB for peroxidase), the influences of the metal ions used in EM were tested on the appearance of the coloured reaction products. Cerium ions used in EM for detection of AcP-ase^Ce activity have been shown to influence the PO^DAB visibility in LM and EM experiments. From the AS reaction media components neither barium ions nor p-nitro catachol sulphate influenced the LM visibility of the PO reaction.     

A dual-immunocytochemical method to localize c-fos protein in specific neurons based on their content of neuropeptides and connectivity

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 m in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP). Secondly, to identify activated neurons in female Syrian hamsters at the time of the proestrous luteinizing hormone surge, c-fos protein staining with DAB was carried out in combination with BDHC staining for gonadotrophin-releasing hormone (GnRH). In both these studies, cells which co-localized the peptide and c-fos protein in the nucleus could be identified unequivocally. Thirdly, to analyse projections of c-fos-immunoreactive neurons, the retrograde tracer, cholera toxin subunit B (ChB) was pressure-injected into the piriform cortex of rats, which were thereafter fully kindled in the contralateral amygdala. The tract tracer was stained with BDHC as the chromogen. Due to the advantages of the dual-labelling methodology, the combination of retrograde tracing and c-fos protein histochemistry provides an excellent method for identifying projecting and activated neurons in the same section.     

Ultrastructural electron probe X-ray microanalytical reaction product identification of three different enzymes in the same mouse resident peritoneal macrophage

Simultaneous cytochemical enzyme localization procedures for peroxidase (PO) plus acid phosphatase (AcP-ase) and/or aryl sulphatase (AS) have been investigated at the ultrastructural (EM) level. Electron probe X-ray microanalysis (EPMA) will identify and differentiate the reaction products. Dual reaction product localization of PO plus AcP-ase or alternatively PO plus AS have been obtained in the same mouse resident peritoneal macrophage. This has been acquired by first performing a PO-reaction followed by AcP-ase or followed by AS. In both cases PO-related reaction products (PODAB/Os or PODAB/Pt) were localized in nuclear envelope (NE) and rough endoplasmic reticulum (RER). Cells were identified by this reaction product as resident macrophages. Reaction products from the AcP-ase related cerium (AcP-aseCe), localized in lysosomes have been identified and differentiated from the PO-related osmium containing products. Similarly AS related barium (ASBa), localized in lysosomal structures and (R)ER was identified and differentiated. Triple reaction product localization of PO followed by AcP-ase plus AS could also be obtained. In this case, PO-related platinum containing reaction products (PODAB/Pt or PODAB/Os) in NE and RER has been identified and differentiated from the AcP-ase related lysosomal cerium (AcP-aseCe) and the AS related barium localized in lysosomal and (R)ER structures. Reversing the sequences in both dual cytochemical procedures: AcP-aseCe or ASBa followed by PODAB/Os (or PODAB/Pt) resulted in AcP-aseCe or ASBa activity related reaction products only. Reversing the sequence in the triple reaction procedures (ASBa followed by AcP-aseCe) resulted in the absence of the barium containing reaction products. By application of OsO4 postfixation with aminotriazole (ATR) additives the detrimental effects upon the various precipitates have been confirmed. In LM studies, using rat intestine and non-metal identification reactions for two of the enzymes (pararosaniline for AcP-ase, DAB for peroxidase), the influences of the metal ions used in EM were tested on the appearance of the coloured reaction products. Cerium ions used in EM for detection of AcP-aseCe activity have been shown to influence the PODAB visibility in LM and EM experiments. From the AS reaction media components neither barium ions nor p-nitro catachol sulphate influenced the LM visibility of the PO reaction.     

Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.     

An ultrastructural double-labelling method: immunohistochemical localization of cell adhesion molecule L1 on HRP-labelled developing corticospinal tract axons in the rat

A double electronmicroscopical (EM) staining was developed which enabled the ultrastructural localization of cell adhesion molecules on the outer axonal membrane of horseradish peroxidase (HRP)-labelled axons in the developing central nervous system (CNS). HRP was used to anterogradely trace outgrowing corticospinal tract (CST) axons in ten-day-old rats. After visualization of HRP using tetramethylbenzidine (TMB) as a chromogen and ammoniumheptamolybdate (AHM) as a stabilizer at pH 6.0 as described previously (Joosten et al. 1987, J Histochem Cytochem 35: 623-626) an additional diaminobenzindine (DAB)-Ni incubation was carried out for further stabilization. Subsequently a preembedding immunoperoxidase (DAB) staining was executed for detection of cell adhesion molecule L1. Using this procedure anterogradely HRP-labelled CST axons were recognizable by a granular black TMB-AHM-DABNi reaction product at the light microscopic (LM) level, which clearly contrasts to the relatively homogeneous brown L1-immunostaining. Electronmicroscopically HRP-labelled CST axons were characterized by the presence of an intracellular crystaloid TMB-AHM-DABNi reaction product which made identification of CST axons rather easy, whereas the L1-DAB precipitate could be noted on the outer axonal membrane of the HRP-labelled CST axons, marking the presence of the L1 cell adhesion molecule. In addition the procedure described in this report preserves ultrastructural details of developing neural tissue. In conclusion, the method presented can be employed in combined HRP-tracing and immunohistochemical electronmicroscopic studies.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postosmication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat

Dipeptidyl peptidase II (DPP II) in normal rat lung was evaluated by the enzymes' ability to hydrolyze Lys-Ala or Lys-Pro derivatives of 4-methoxy-2-naphthylamine (MNA). For visualization of this activity, the liberated MNA was coupled with fast blue B for light microscopy (LM) or hexazotized pararosaniline with osmication for electron microscopy (EM). Granular to diffuse reaction product was noted in many lung cells in frozen sections for LM, including alveolar and tissue macrophages, fibroblasts, chondrocytes, bronchial and bronchiolar epithelial cells and mast cells. Reaction product at the EM level was seen in the lysosomal structures of the above cells, although lysosomal heterogeneity with regard to reactivity was noted. Cellular content of reaction product by EM correlated with LM staining intensity. Additional structures, not obviously reactive by LM, such as the lamellar bodies of type II cells and lysosomes in other cell types, were seen to contain reaction product ultrastructurally. A modified biochemical assay for the quantitation of DPP II in tissue homogenates was used to determine the activity of the enzyme in rat lung. Enzyme activity in polyacrylamide isoelectric focusing gels indicate that Lys-Ala-MNA was the more specific substrate but, by virtue of its rapid hydrolysis, Lys-Pro-MNA was more sensitive.     

An ultrastructural double-labelling method: immunohistochemical localization of cell adhesion molecule L1 on HRP-labelled developing corticospinal tract axons in the rat

Summary A double electronmicroscopical (EM) staining was developed which enabled the ultrastructural localization of cell adhesion molecules on the outer axonal membrane of horseradish peroxidase (HRP)-labelled axons in the developing central nervous system (CNS). HRP was used to anterogradely trace outgrowing corticospinal tract (CST) axons in ten-day-old rats. After visualization of HRP using tetramethylbenzidine (TMB) as a chromogen and ammoniumheptamolybdate (AHM) as a stabilizer at pH 6.0 as described previously (Joosten et al. 1987, J Histochem Cytochem 35:623–626) an additional diaminobenzindine (DAB)-Ni incubation was carried out for further stabilization. Subsequently a pre-embedding immunoperoxidase (DAB) staining was executed for detection of cell adhesion molecule L1. Using this procedure anterogradely HRP-labelled CST axons were recognizable by a granular black TMB-AHM-DABNi reaction product at the light microscopic (LM) level, which clearly contrasts to the relatively homogeneous brown L1-immunostaining. Electronmicroscopically HRP-labelled CST axons were characterized by the presence of an intracellular crystaloid TMB-AHM-DABNi reaction product which made identification of CST axons rather easy, whereas the L1-DAB precipitate could be noted on the outer axonal membrane of the HRP-labelled CST axons, marking the presence of the L1 cell adhesion molecule. In addition the procedure described in this report preserves ultrastructural details of developing neural tissue. In conclusion, the method presented can be employed in combined HRP-tracing and immunohistochemical electronmicroscopic studies.     

New Silver-Gold Intensification Method of Diaminobenzidine for Double-Labeling Immunoelectron Microscopy

The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3′-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.     

Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immobilized on nitrocellulose

The nitrocellulose model and microphotometry were used to investigate whether in immunoperoxidase cytochemical methods the amount of final reaction product reflects the amount of cell surface antigen. The results obtained with four cytochemical peroxidase methods, i.e., those using diaminobenzidine/H2O2 (DAB/H2O)2, DAB/H2O2/COCl2, DAB/H2O2/imidazole, and silver intensification of the DAB end product, were compared first. The quantitative DAB/H2O2/imidazole method proved to be the most sensitive and was selected for further studies. Cell surface antigens prepared by solubilization of peritoneal macrophages with octyl-beta-D-glucopyranoside were immobilized on nitrocellulose. Monoclonal antibody binding to these cell antigens was detected by peroxidase immunocytochemistry. Comparison of the sensitivity of the indirect immunoperoxidase and the biotin-(strept)avidin immunoperoxidase methods on the basis of the highest detectable dilution of a cell lysate showed that these methods were equally sensitive. A linear relationship between the absorbance of the peroxidase reaction product and the amount of cell lysate immobilized on nitrocellulose was found for all three indirect immunoperoxidase methods. This proves that the amount of final immunocytochemical peroxidase reaction product is proportional to the amount of antigen in cell lysates. However, the relative expression of antigens in intact cells differs from that in cell lysates. Therefore, the present method to solubilize cells and immobilize cell antigens cannot be used to quantitate the antigen content of cells.     

Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.

We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)     

Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases

The reaction product of cerium used for localization of peroxisomal oxidases is highly electron-dense but lacks sufficient contrast at the light microscopic level. We describe two methods for converting the reaction product of cerium to colored compounds visible by light microscopy. The first method is based on 3,3'-diaminobenzidine (DAB) amplification of transition metal compounds, of which cerium is one. Sections of glutaraldehyde-fixed rat liver or kidney are incubated first in media for various oxidases containing CeCl3, followed by treatment with DAB in Na acetate buffer, pH 5.3. To prevent any interference by the peroxidatic activity of catalase, NaN3 or Na pyruvate is added to the DAB amplification medium. Staining with DAB can be further intensified with NiCl2 or CoCl2. The second method is based on the conversion of the cerium reaction product with alkaline lead citrate and the final visualization of the lead compound with ammonium sulfide. These methods allow the evaluation of large sections for peroxisomal oxidases by light microscopy, making close correlation between light and electron microscopy possible.     

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3''-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.     

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post-embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.     

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.     

Immunocytochemical localization of nuclear antigens in the unicellular green alga,Chlamydomonas reinhardtii,processed by cryofixation and freeze-substitution

Summary We describe the preparation of monoclonal antibodies to nuclear antigens in the green alga,Chlamydomonas reinhardtii, and their localization at the light and electron microscope level. Supernatants from hybridomas were screened by the ELISA method and the four antibodies giving the strongest signal were subjected to further analysis. At the LM level immunogold silver staining was used on semi-thick resinless sections. We have examined at the EM level the distribution of these antigens by post-embedding immunocytochemical techniques on sections of conventionally fixed specimens compared to cryofixed and freeze-substituted ones. Enhanced ultrastructural preservation was observed in cells which were cryofixed, freeze-substituted and embedded at –35°C in Lowicryl K4M. Different preparative procedures involving cryofixation and substitution are described. Of the four antibodies three were localized under light and electron microscopy. All three were distributed in the interchromatin space. One of these antigens (QUL4D2, 54 kDa) is also found in the dense fibrillar component and fibrillar centers of the nucleolus.Abbreviations DFC dense fibrillar component - EM electron microscope - FC fibrillar center - GAM5 goat anti-mouse IgM coupled to 5 nm colloidal gold - Ig immunoglobulin - LM light microscope - MAb monoclonal antibody - PAG protein A-gold - PBS phosphate buffered saline - PEG polyethylene glycol     

Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles.

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.