Application of Dual Pre-Embedding Stains for Gonadotropins to Pituitary Cell Monolayers with Avidin-Biotin (ABC) and Peroxidase-Antiperoxidase (PAP) Complexes: Light Microscopic Studies

Double stains for gonadotropins and gonadotropin-releasing hormone were developed for fixed whole pituitary cells from cycling female rats. Monolayer cells were stimulated with [d-Lys⁶]GnRH, fixed in 2.5% glutaraldehyde, and then stained for luteinizing hormone (LH) (1:50,000-12 h) or follicle stimulating hormone (FSH) (1:60,000-12 h) and the avidin-biotin-peroxidase complex technique (ABC) with a jet-black substrate (nickel intensified diaminobenzidine—DAB). This was followed by a stain for the other gonadotropin with either ABC or peroxidase-antiperoxidase complex (PAP) techniques and amber (DAB) or red (3-amino-9-ethyl-carbazole) substrates. Additional monolayers were stimulated with biotinylated [d-Lys⁶]GnRH and stained with the ABC technique and the black (nickel-DAB) substrate. These monolayers were then stained immunocytochemically for LH or FSH with either ABC or PAP methods and orange or red substrates. The controls showed that the omission of the second primary antiserum abolished the stain indicating that the second staining solutions did not react with components in the first group. The addition of the second peroxidase substrate in sequence after the first stain indicated that no residual peroxidase activity remained from the first stain. Our tests also showed that saponin was not needed to aid reagent or antibody penetration. The dual stains demonstrated that 30-60% of the gonadotropes stored LH and FSH together, often in separate regions of the same cell. Some cells contained only one hormone (20-22%). The dual stains for GnRH and gonadotropins demonstrated that 80-90% of the GnRH bound cells are gonadotropes. These techniques allow a study of storage sites for multiple hormones in or on whole cells. The studies agree with and augment the results from the use of serial sections.     

Application of a rapid avidin--biotin--peroxidase complex (ABC) technique to the localization of pituitary hormones at the electron microscopic level

The new avidin--biotin--peroxidase complex (ABC) technique was applied to ultrathin sections of rat pituitary that were fixed with glutaraldehyde and embedded in Araldite 6005. The primary antisera dilutions that are normally applied for 24-48 hr with the peroxidase-antiperoxidase (PAP) complex technique were used. High background was observed with the ABC method when incubation times were 12-48 hr. Tests were then conducted with shorter incubation times. The staining intensity was measured with a densitometer. Detectable stain was seen after only 15 min in dilutions of 1:10,000 anti-bovine luteinizing hormone (bLH beta), 1:8000 anti-rat thyroid-stimulating hormone (rTSH beta), and 1:20,000 anti-25-39-adrenocorticotropic hormone (25-39ACTH). Optimal LH staining was seen after 30 min, whereas optimal staining for TSH or ACTH required 1 hr. Stain was detectable with a dilution of 1:4000 anti-human follicle-stimulating hormone (hFSH beta) after 30 min and was optimal after 4 hr. Prolonged incubation times with these dilutions decreased the staining intensity because a deposit of high background was produced that appeared as a filigreed network over the cells. When higher dilutions were tested with 2-hr incubation times, optimal staining was seen with 1:30,000 anti-bLH beta, 1:24,000 anti-rTSH beta, 1:30,000 anti-25-39ACTH, and 1:8000 anti-hFSH beta. These tests demonstrate the potential of the ABC method for the rapid detection of small amounts of specific and nonspecific antibodies that are bound to pituitary cells.     

Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures

The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.     

Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies

The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best immunohistological labeling reactions, being stable on storage and compatible with a wide range of human monoclonal antibodies. Gel filtration revealed that monoclonal PAP is of lower molecular weight than conventional PAP complexes (fulfilling theoretical predictions based on the monospecificity of monoclonal antibodies).     

NADH-dehydrogenase reaction in combination with immunoperoxidase (PAP) staining for light microscopic observation on the interneuronal relations of the enteric nervous system of the pig

A novel procedure for a simultaneous demonstration of particular enteric nerve cell types and peptidergic nerve fibres has been developed by combining the histochemical reaction for NADH-dependent dehydrogenase and the unlabelled antibody peroxidase-antiperoxidase (PAP) method described by Sternberger. Whole-mount spreads were successively incubated in a NADH: nitroblue tetrazolium solution, fixed with a picric acid/formaldehyde mixture, dehydrated, cleared and rehydrated before processing for immunocytochemical localization of the neuropeptide by the PAP method. The nerve cells appear heavily stained by deposits of dark blue formazan, whereas the peptide-containing nerves appear bright brown. In the myenteric and submucous plexuses of the porcine small intestine the devised method allows an appropriate identification of Dogiel's type I, type II and type III neurons surrounded by varicose enkephalin-like immunoreactive fibre baskets with button-like twigs to the very surface of the ganglionic cells, suggestive of synaptic connections.     

Relation between ultrastructural presence of CEA using peroxidase-antiperoxidase (PAP) method and tissue and serum levels of CEA in patients with breast cancer.

Ultrastructural studies were performed to detect the presence of carcinoembryonic antigen (CEA) in breast cancer by peroxidase-antiperoxidase (PAP) method. The evidenced presence of CEA was compared with the serum and tissue concentrations. A correlation between the presence of CEA at the ultrastructural level and tissue concentration was observed but not with serum levels. These studies revealed positive immunocytochemical staining for CEA when antigen concentration was 700 ng/g tissue and the reaction was strongly positive when the concentration was greater than or equal to 2000 ng/g tissue.     

Localization of receptor-human chorionic gonadotropin complex to the surface of the luteal and interstitial glandular cells in the pseudopregnant rat ovaries with the peroxidase-antiperoxidase (PAP) complex method

Receptor-bound hCG was localized in pseudopregnant rat ovarian cells at semiultrastructural level with the peroxidase-antiperoxidase (PAP) complex method. The animals received 2, 6, 12 or 24 h prior to killing a single intravenous injection of hCG and the hormone was localized in the 5-micrometer paraffin sections and in the 1-micrometer epon sections using the pre-embedding technique. The peroxidase staining localized to the periphery of the luteal and interstitial glandular cells. No significant staining occurred in the intracellular structures of the cells during the 24-h observation period. However, the appearance of staining in the subplasmalemmal structures can not be excluded. These results are compatible with the previous observations that the receptor-hCG complexes are primarily formed at the surface of the luteal and interstitial glandular cells.     

Localization of receptor-human chrionic gonadotropin complex to the surface of the luteal and interstitial glandular cells in the pseudopregnant rat ovaries with the peroxidase-antiperoxidase (PAP) complex method

Summary Receptor-bound hCG was localized in pseudopregnant rat ovarian cells at semiultrastructural level with the peroxidase-antiperoxidase (PAP) complex method. The animals received 2, 6, 12 or 24 h prior to killing a single intravenous injection of hCG and the hormone was localized in the 5-m paraffin sections and in the 1-m epon sections using the pre-embedding technique. The peroxidase staining localized to the periphery of the luteal and interstitial glandular cells. No significant staining occurred in the intracellular structures of the cells during the 24-h observation period. However, the appearance of staining in the subplasmalemmal structures can not be excluded. These results are compatible with the previous observations that the receptor-hCG complexes are primarily formed at the surface of the luteal and interstitial glandular cells.This work was supported by a grant from The Academy of Finland     

Self-assembled monolayers of bis(salicylaldiminato)nickel(II) Schiff-base complexes: synthesis and structure

The synthesis of two derivatized Ni^II Schiff-base complexes is reported. Self-assembled monolayers (SAMs) have been obtained by reaction of coupling layers, functionalized glass substrates and the derivatized complex precursors. The self-assembled films have been characterized by contact angle measurements, X-ray photoelectron spectroscopy, and UV-Vis absorption spectroscopy. A structure of these SAMs is proposed on the basis of spectroscopic data and molecular metrical parameters.     

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macroanatomic,light microscopic,and scanning electron microscopic studies of the tongue in the seagull (Larus fuscus) and common buzzard (Buteo buteo)

The tongues of ten seagulls and six common buzzards were examined. In both species, papillae linguales caudales were shaped like a letter “V” between the corpus linguae and the radix linguae. From these papillae, the length of the laterally placed papillae was greater compared with others in both species. Two or three secondary papillae were detected on these papillae in the seagull. In scanning electron microscope (SEM) examinations, in the seagull, the apex linguae was composed of multilayered desquamated cells, while in the buzzard, scalelike simple projections on the surface of desquamated cells were observed. In the buzzard, glandula (gll). linguales, and gll. mandibulares caudales were seen, while in the seagull, gll. cricoarytenoideae and gll. mandibulares caudales were present. In the seagull, apex linguae were bifurcated, and there were desquamating multilayered cells, particularly at the apex linguae. The number and location of salivary gland orifices are specific to this species. The common buzzard had similarities to many characteristics of the long‐legged buzzard. An absence of long and curly threadlike projections at the two lateral sides of the corpus linguae and an excessive number of salivary gland orifices at the corpus linguae were the main differences from the long‐legged buzzard.     

Chemical, biological and immunological properties of pituitary gonadotropins from the donkey (Equus asinus): comparison with the horse (Equus caballus)

Donkey gonadotropins (donkey luteinizing hormone, dLH; donkey follicle-stimulating hormone, dFSH) have been isolated in purified form from 191 donkey pituitaries using essentially the same procedures previously employed for the purification of equine gonadotropins. Chemically, dLH and dFSH were observed to be similar to equine LH (eLH) and FSH (eFSH) in fractionation behavior and glycoprotein nature. Two forms of the dFSH molecule were observed, as is the case for eFSH. Donkey LH had significantly less total carbohydrate (13.5%) and sialic acid (1.9%) than eLH (26.7% and 5.8%, respectively). Carbohydrate (17-21%) and sialic acid (2.4%) content of the two dFSH preparations closely resembled that of eFSH. A slightly higher tyrosine content in the donkey gonadotropins was noted in a comparison of amino acid compositions. Immunologically, in a heterologous FSH radioimmunoassay (RIA), dFSH preparations were equal to or twice as active as eFSH preparations. However, in homologous RIAs for equine chorionic gonadotropin (eCG), eFSH and eLH, both the dLH and dFSH preparations were considerably less active than the equine gonadotropins, and their inhibition curves were all nonparallel. Biologically, in the Steelman-Pohley assay both dFSH preparations were equipotent and as potent as eFSH (approximately 40 times NIH-FSH-S12). In the Sertoli cell assay for cAMP (FSH assay) and the Leydig cell assay for testosterone (LH assay), both dFSH and dLH were 2- or 6-fold more active than eFSH and eLH, respectively. In rat and equine testis FSH homologous radioreceptor assays, dFSH preparations were as active and up to 6-fold more active than eFSH. In contrast, dLH was 10-fold less active than eLH in the equine LH homologous radioreceptor assay. Unlike eLH, dLH was found to possess little intrinsic FSH activity or FSH inhibitory activity, and the small amount of FSH activity observed was most likely due to FSH contamination. Therefore, eLH behaves much like eCG (pregnant mare's serum gonadotropin, PMSG) which also possesses both LH and FSH activity. In contrast, dLH behaves more like donkey chorionic gonadotropin (dCG) which possesses only a low degree of FSH activity.     

A comparison of the avidin-biotin-peroxidase complex (ABC) and peroxidase-anti-peroxidase (PAP) immunocytochemical techniques for demonstrating Sendai virus infection in fixed tissue specimens

Mice were infected with Sendai virus and killed 8 days later. Lungs were removed and perfused with ethanol, 10% neutral formalin, Bouin's, B-5, or Zenker's fixatives. Tissues were dehydrated, embedded in paraffin, sectioned and stained for the presence of Sendai virus using the avidin-biotin-peroxidase-complex (ABC) and peroxidase antiperoxidase (PAP) immunocytochemical techniques. Results of these techniques were compared. The ABC technique was more sensitive than the PAP. Sendai antigen was demonstrated by the ABC technique in lung tissue fixed with any fixative, whereas antigen could be demonstrated with consistency only in ethanol-fixed lung by the PAP technique. Trypsin treatment of lung prior to immunoperoxidase treatment failed to enhance staining with either technique and actually caused a decrease in staining in ethanol, B-5 and Zenker's-fixed specimens.     

Quasielastic light scattering,electrophoresis, and fluorescence studies of lysozyme-poly(2-acrylamido-methylpropylsulfate) complexes

Complexation between lysozyme and sodium poly(2-acrylamido-2-methylpropanesulfonate) (PAMPS) was studied by quasielastic light scattering, electrophoretic light scattering, fluorescence, and turbidimetry in electrolyte solution. These techniques show that complexation occurs at pH 9.6 in an ionic strength buffer of 0.25M NaCl + 25 mM Na₂B₄O₇. At constant lysozyme concentration (C_pro). The structure of the complex depends on the polymer concentration. At low polymer concentration (relative to C_pro), an intrapolymer complex is formed. This intrapolymer complex aggregates to an interpolymer species upon increase in polymer concentration. Complex formation was also studied by fluorescence using pyrene-labeled PAMPS (Py-PAMPS). Energy transfer from singlet-excited tryptophan residues in lysozyme to the pyrene label occurs when the complex is formed. Fluorescence and turbidity data indicate that lysozyme interacts with Py-PAMPS preferentially at pyrene sites, which leads to static quenching of tryptophan fluorescence via energy transfer to the pyrene label. © 1995 John Wiley & Sons, Inc.     

Partial urethral obstruction enhances NADPH-diaphorase activity in the monkey (Macaca fascicularis) bladder: light and electron microscopic studies

The effect of partially obstructing the urethra on the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) bladder was examined by light and electron microscopy. Partial urethral ligation was done in adult male monkeys. The animals were sacrificed 2, 4 weeks after partial urethral obstruction. This was compared to controls (normal and sham operated). Urethral obstructed animals were observed to have increased urinary frequency and decreased urinary flow rate. Two weeks after urethral obstruction, the overall NADPH-d activity in the intramural ganglia of the bladder base was enhanced compared to control animals. The frequency of intensely stained NADPH-d positive neurons was increased compared to the control animals. About one-third of intensely stained NADPH-d positive neurons appeared to undergo degenerative changes. At 4 weeks after urethral obstruction, a wide occurrence of NADPH-d positive neurons in advanced stages of degeneration in the bladder base was observed. Cellular debris was strewn among normal looking ganglion cells and along the nerve processes. The proportion of intensely stained NADPH-d positive neurons was relatively lower than the controls. The total number of NADPH-d positive neurons and the nerve fibres in the entire bladder was significantly reduced when compared to control animals. Electron microscopy showed some NADPH-d activity in intramural ganglion cells in 2 weeks after partial urethral obstruction. NADPH-d reaction product (formazan) was deposited on the membranes of the rough endoplasmic reticulum, and the outer membranes of some mitochondria in the intramural neuron. At 4 weeks after urethral obstruction, NADPH-d was present in the membrane of the mitochondria and some mitochondria appeared swollen with disrupted cristae. Present results show that NADPH-d activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) urinary bladder was increased after two weeks and reduced after 4 weeks of partial urethral obstruction. It is speculated that the increased NADPH-d activity associated with partial urethral obstruction would lead to neuronal damage and death, which may contribute to detrusor overactivity. However, it warrants further investigation to understand the mechanism of neuronal cell death after partial urethral obstruction.     

Immunocytochemical localization of fibronectin (LETS proteins) on the surface of L6 myoblasts: light and electron microscopic studies.

Fibronectin (LETS protein) is a major cell surface glycoprotein component of a variety of nontransformed, substrate-attached cells in culture. Its presence has been related to increased adhesive properties. Using the peroxidase-antiperoxidase method to localize antibodies to fibronectin, we have observed that the distribution of fibronectin on L6 myoblasts varies with the density of the culture and the differentiative state of the cells. Low density, undifferentiated cultures of L6 myoblasts have a sparse accumulation of fibronectin; the antibody-antigen reaction indicates its presence on cell membranes, especially where several cells are in proximity. Undifferentiated cells in high density cultures have two forms of fibronectin localization-a diffuse staining on the membrane and a dense staining on an extracellular filamentous matrix. This matrix is composed of filaments ranging from 20–25 nm in diameter which occur singly or coalesce to form bundles. The filaments in this matrix are also observed to have dense globules scattered along their length. These filaments, which are at least in part composed of fibronectin, also react with concanavalin A, as do certain plasma membrane components. In contrast to the observations seen in undifferentiated cells, differentiated cells or myotubes have a diffuse membrane staining with antifibronectin antibodies, and the filamentous form is usually absent.     

Electron microscopic and physico-chemical studies of DNA complexes with synthetic oligopeptides: binding specificity and DNA compact structures

Binding to DNA of two synthetic peptides, Val-Thr-Thr-Val-Val-NH-NH-Dns and Thr-Val-Thr-Lys-Val-Gly-Thr-Lsy-Val-Gly-Thr-Val-Val-NH-NH-Dns (where Dns is a residue of 5-dimethylaminonaphthalene-1-sulfonic acid), has been studied by circular dichroism, electron microscopy and fluorescence methods. It has been found that these two peptides can self-associate in aqueous solution as follows from the fact that concentration-dependent changes are observed in the UV absorbance and fluorescence spectra. The two peptides can bind to DNA both in self-associated and monomeric forms. The pentapeptide in the beta-associated form binds more strongly to poly(dG).poly(dC) than to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) whereas the tridecapeptide exhibits an opposite order of preferences binding more strongly to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) than to poly(dG).poly(dC). Binding is a cooperative process which is accompanied by the DNA compaction at peptide/DNA base pair ratios greater than 1. At the initial stage of the compaction process, the coalescence of DNA segments covered by bound peptide molecules leads to the formation of DNA loops stabilized by the interaction between peptide molecules bound to different DNA segments. Further increase in the peptide/DNA ratio leads to the formation of rod-like structures each consisting of two or more double-stranded DNA segments. The final stage of the compaction process involves folding of fibrillar macromolecular complexes into a globular structure containing only one DNA molecule.     

DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes:

Two complexes of [Co(phen)2IP]3+ (IP=imidazo[4,5-f][l,10]phenanthroline) and [Co(phen)2PIP]3+ (PIP=2-phenylimidazo[4,5-f][1,10]phenanthroline) have been synthesized and characterized by UV/VIS, IR, EA and mass spectra. The binding of the two complexes with calf thymus DNA has been investigated by absorption spectroscopy, cyclic voltammetry, viscosity measurements and DNA cleavage assay. The spectroscopic studies together with cyclic voltammetry and viscosity experiments support that both of the complexes bind to CT DNA by intercalation via IP or PIP into the base pairs of DNA. [Co(phen)2PIP]3+ binds more avidly to CT DNA than [Co(phen)2IP]3+, which is consistent with the extended planar and pi system of PIP. Noticeably, the two complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA.