Electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of the calf fundic glands

The electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of calf fundic glands was studied using specific antisera for prochymosin and pepsinogen with a protein A-gold method. Prochymosin and pepsinogen immunoreactivities were detected in the same secretory granules of the chief, mucous neck and transitional cells, simultaneously using small and large colloidal gold particles. In chief cells, both immunoreactivities were distributed uniformly over the same zymogen granules showing a round, large, homogeneous and electron-dense appearance. In mucous neck cells, both immunoreactivities were found exclusively on the same electron-dense core located eccentrically in the mucous granule showing light or moderate electron density. In transitional mucous neck/chief cells, electron-dense cores became larger in size and some granules were occupied by the electron-dense core without a halo between the core and the limiting membrane. Both immunoreactivities were found uniformly over the electron-dense core. The granules having no halo in the transitional cells could not be distinguished from the typical zymogen granules in the chief cells.     

Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections.

ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cells was identified as containing numerous round or ovoid 170--300 nm secretory granules.     

Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections

Summary ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cell was identified as containing numerous round or ovoid 170–300 nm, secretory granules.     

Aurosomes produced in the synovial membrane by the oral administration of a gold compound SK & F 36914.

Chlorotriethylphosphine gold (SK & F 36914) administered orally to rabbits produced aurosomes in the synovial membrane. These aurosomes were similar to aurosomes produced by parenteral or intra-articular injections of soluble gold salts such as sodium aurothiomalate and aurothioglucose. This study shows, (1) that an orally administered gold compound is capable of producing gold deposits in the synovial membrane, and (2) that such compounds may have a future in the treatment of rheumatoid arthritis.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.     

Endocytosis in cultures of Blastocystis hominis

A study of the function of the electron-dense pits in the vacuolar and granular forms of Blastocystis hominis was undertaken. Immuno-electron microscopy using anti-clathrin antibody and colloidal gold demonstrated clathrin to be associated with all forms of the pits and some cytoplasmic vesicles. Cationized ferritin traced the pathway of endocytosis from the surface of the coated pits through internalization via electron-dense coated vesicles and uncoated vesicles and tubules in the cytoplasm. The cationized ferritin particles accumulated in the central vacuole, suggesting a metabolic or storage role for this structure. Differences in the accumulation of cationized ferritin particles were noted between vacuolar and granular forms. The hydrolytic enzyme acid phosphatase was not detected within the central vacuole suggesting that this structure does not act as a lysosome.     

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Long term effects of Myochrysine in articular cartilage

Intra-articularly administered sodium aurothiomalate (Myochrysine) produced aurosomes containing characteristic electron dense contents (indicating the presence of gold), in the chondrocytes of rabbit articular cartilage. At first the aurosomes were bounded by a membrane but later the electron dense contents were seen lying free in the cytoplasmic matrix. Such deposits were detectable up to 14 months after injection of Myochrysine but none were found at later time intervals (18 months and 2 years). There was a reduction in the population of superficial chondrocytes (Zone I) while those in deeper zones (Zones II and III) showed an increased content of intracytoplasmic filaments. It is thought that these are regressive or degenerative changes produced by gold.     

Direct detection of immunogold reactions by real-time video microscopy

Summary Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

Direct detection of immunogold reactions by real-time video microscopy.

Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.     

Intragranular colocalization of immunoreactive methionine-enkephalin and oxytocin within the nerve terminals in the posterior pituitary

To determine differential tissue antigens in the same section immunocytochemically using the electron microscope, the neurohypophysis was examined following the application of a freeze-drying tissue preparation and staining with the protein A-colloidal gold-antibody complex method (Hisano S, Adachi T, Daikoku S: J Histochem Cytochem 32:705, 1984). At the light microscopic level, colocalized immunostaining for methionine-enkephalin (ENK) and oxytocin (OXT) was found in the rat neurohypophysis under different physiological states. Small pieces of the neurohypophysial tissue were frozen and dried. The dried tissue was fixed with paraformaldehyde vapor and embedded. The ultrathin sections were stained with the antibody for ENK coupled with protein A-small colloidal gold, and antibody for OXT or vasopressin (VP) conjugated with protein A-large colloidal gold. The ultrastructures of the nerve terminals were well preserved and showed many membrane-limited secretory granules. It was possible to identify both OXT- and VP-containing nerve terminals as their secretory granules were differentially labeled with protein A-colloidal gold anti-OXT or anti-VP complex, respectively. The secretory granules, which were labeled with large gold particles for OXT, also carry small gold particles. It is evident that ENK coexists with OXT in the same granules.     

Protein-gold complexes as neuronal markers for long-term tracing studies

In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.     

Light microscopic localization of silver-enhanced liposome-entrapped colloidal gold in mouse tissues

Silver-enhanced liposome-entrapped colloidal gold was developed for light microscopic localization of liposomes. Preparation of colloidal gold entrapped in liposomes was achieved by a modified method of Hong, et al. (1983) Biochim. Biophys. Acta 732, 320-323). In this report, a gold chloride/citrate solution of low pH (3.4) was used to inhibit the formation of gold granules during the liposome preparation. The diameter of most liposomes ranged from 80 to 100 nm. Following liposome preparation, the pH was adjusted to 6, and the temperature increased to 55 degrees C. The majority of the liposomes contained one to three gold particles. Liposomes were injected into mice via tail vein; 24 h later, tissues were collected. Sections were processed for silver enhancement of the gold particles and examined by light microscopy. Silver-enhanced gold particles were clearly observed in both liver and implanted tumor. Localization was confirmed by electron and fluorescence microscopy. Thus, we have shown that silver enhancement of colloidal gold liposomes is a direct and sensitive method for tracing the fate of liposomes in vivo, providing minimal background interference and a good definition of various cell types.     

Comparison of detecting sensitivities of different sizes of gold particles with electron-microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model

The detecting sensitivities of different-sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha-ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 micron 2 nongranule area from that in 1 micron 2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha- and beta-ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha- and the beta-ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.     

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Protein-gold complexes as neuronal markers for long-term tracing studies

Summary In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.     

Ultrastructural localization of relaxin in the corpus luteum of the pregnant and early lactating tammar wallaby, Macropus eugenii

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200–350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition. Received: 3 March 1997 / Accepted: 26 May 1997     

Alcohol abuse-related mesangial glomerulonephritis: immunoelectron microscopy

Immunoelectron microscopy, using post-embedding immunohistochemistry with colloidal gold, was performed on renal samples from forensic autopsies. We confirmed that electron-dense deposits seen in alcohol abuse-related mesangial nephritis correspond to immunoglobulins, as has been shown previously by others in idiopathic cases. We investigated seven control samples and 13 specimens from individuals with evidence of alcohol abuse, six of whom had mesangial nephritis with IgA deposition. We found concentration of the gold particles over large electron-dense deposits in four of six cases of mesangial nephritis, confirming that they correspond to the IgA shown by immunofluorescence. Furthermore, a similar concentration of gold particles was not observed in control cases or in alcoholics without mesangial glomerulonephritis (4/6 vs 0/14; p = 0.005). IgM, seen as small aggregates, was confirmed in only two of six of the same cases. This is the first time that immunoelectron microscopy is performed on tissues obtained post mortem.