Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

Summary The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB-and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.Supporied by grants from the Ministry of Education, Science and Culture of Japan     

Combined technique of immunohistochemistry and autoradiography for the simultaneous detection of steroid hormone receptors and their ligand

A combined technique of immunohistochemistry and autoradiography was applied to detect progesterone target cells in tissue sections. The radioactively labeled progestin [3H]-Organon 2058 and the progesterone receptor, revealed by antibodies to the receptor molecule, were localized simultaneously in identical cells on the same tissue section. Technical details that make possible combined detection of the nuclear antigen, present in very small amounts, simultaneously with its steroid ligand are described.     

Distribution of LH-RH nerve endings in the median eminence of proestrus female rats: fluorescence and peroxidase anti-peroxidase (PAP) immunohistochemistry.

The morphological distribution of the nerve terminals containing LH-RH in the hypothalamus especially in the median eminence of the proestrus female rat was demonstrated by immunohistochemistry, using FITC and peroxidase antibody. The terminals containing LH-RH were classified into four groups on the topographic relationship. LH-RH nerve processes terminated mainly in the infurdibular radix within an elliptical zone surrounding the bases of the infundibular recessus. The heaviest concentration of LH-RH terminals immunohistochemically demonstrated lay on each side of the region extending from the dorsal part of tuberoinfundibular sulcus to the lateral part of the external layer of the superior labium of the infundibulum. We were unable to detect any neuronal soma with the immunoreactivity to LH-RH in the hypothalamic gray matter. The distributional patterns of LH-RH, GH-RIH and monoamine in the median eminence as well as their relationships were briefly discussed.     

A simple fluorescence staining technique for the differentiation of human tissue transplanted into nude mice.

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during interphase appears as brilliant fluorescent chromocenters. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangements changes according to the stage of differentiation of the cell of the position of a particular nucleus within the cell cycle.     

A post-embedding method: demonstration of fibronectin on human liver by PAP technique

Fibronectin, one of the most relevant components of extracellular matrix, seems to mediate cell to cell and cell to substrate interactions by means of selective links with collagen fibrils and glycosaminoglycans. Post-embedding technique using PAP method has allowed us a precise localization of fibronectin on semi-thin sections and on adjacent thin sections, improving the knowledge of fibronectin-collagen relationships.     

Polyester wax embedding and sectioning technique for immunohistochemistry

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.     

Serotonin-storing cells of the chicken duodenum: light,fluorescence and electron microscopy,and immunohistochemistry

Summary In an attempt to identify duodenal endocrine cells emitting formaldehyde-induced fluorescence (FIF), chicken duodena were studied by combined fluorescence, ultrastructural, silver impregnation and immunohistochemical methods in the same or consecutive sections. Our results show that: (1) Almost all the cells emitting yellow fluorescence by both the Falck-Hillarp and the Furness methods exhibit an immunohistochemical reaction with serotonin (5-HT) antiserum. (2) Almost all cells radiating yellow fluorescence by the Furness method stain with toluidine blue in Epon-embedded sections but, by high-voltage electron microscopy, can be subdivided into two types of cell containing either small round or polymorphous types of granules. (3) In the sections from which resin had been removed, all the cells emitting yellow FIF show argentaffinity by the Singh method, but not all cells display argyrophilia with the Grimelius method. (4) Cells exhibiting both argyrophil and argentaffin reactions in deresined serial sections are also separated into two types of cell, containing either small spherical or polymorphous types of granules by conventional electron microscopy in thin sections. Therefore, chicken enterochromaffin cells emit yellow FIF, store 5-HT, show both argentaffinity and argyrophilia, but are ultrastructurally classified into two types of granule-containing cells which may be related to polypeptides coexisting with 5-HT.     

Histamine immunohistochemistry: a new and highly sensitive method for studying cutaneous mast cells

Summary Mast cells have characteristic granulae containing various glucoseaminoglycans, proteases and amines (predominantly histamine). The conventional histological methods for studying mast cells are based upon acidic ortho-and metachromatic routine stains of the glucoseaminoglycans. However, the success of these procedures is dependent upon both the fixatives and the tissues used. In this study, we wanted to find out whether an immunohistochemical procedure could overcome some of these difficulties. Normal human skin was fixed in five different types of fixative and processed for indirect immunofluorescence, using an antiserum to histamine. Only one, 4% carbodiimide in 0.1m phosphate buffer (pH 7.4), resulted in immunostaining. The quality of the staining was good, with a high signal-to-noise ratio, and was located on the mast cells. The method, made it possible to visualize small structures such as a single secreted granula, the thin cytoplasmatic extension of some cells, and a previously undescribed dendritic morphology of some of the mast cells. We therefore recommend, this procedure for cellular studies of mast cells when accuracy is needed.     

Improved technique for the gut fluorescence method in a feeding study of small zooplankton

An Increase in the sensitivity of chlorophyll pigment analysiswas provided by small-volume quartz cuvettes with a specialholder for the determination of gut chlorophyll pigment in anindividual small zooplankton.     

Combined method of PAP immunocytochemistry and autoradiography: application to cell kinetic study in plasma cell dyscrasias

The plasma cell labeling index (LI), in spite of being a reliable indicator for diagnosis and prognosis of multiple myeloma, has been measured in a limited number of laboratories because of technical difficulties. We have developed a new combined technique, using the peroxidase-antiperoxidase (PAP) method and autoradiography, which has several advantages over previously described methods. The primary advantages of our method in the determination of lymphoid-plasma cell LI% are: (a) no damage to slides during storage of more than 1 year; (b) an exact LI measurement in each morphological variety of pleomorphic immunoglobulin-containing cells; (c) no problem in differentiation of lymphoid plasma cells from early red cell precursors; and (d) a separate LI measurement for those lymphoid-plasma cells composed chiefly, if not exclusively, of monoclonal or neoplastic cells. Because of these advantages, this accurate and less difficult technique will facilitate performance of lymphoid plasma cell LI in a number of laboratories.     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

A simplified method for the rapid preparation of peroxidase-anti-peroxidase (PAP) complexes

Summary A modified method is described for the rapid production of peroxidase-antiperoxidase complexes to be used in immunocytochemistry. In this method anti-peroxidase antibodies are precipitated from crude serum with peroxidase at equivalence and subsequently resolubilized at low pH with excess peroxidase. The complexes are isolated from unbound immunoglobulin and peroxidase by gelfiltration. The method combines the advantages of both previously described preparation procedures. The resulting PAP-complex, when tested in indirect immunocytochemistry, is comparable to that obtained in established preparation procedures.Supported by a grant from the Queen Wilhelmina Cancer Foundation (grant no. PA 77/52)     

A method for the synthesis of an oseltamivir PET tracer

A protocol applicable for the synthesis of an oseltamivir positron emission tomography (PET) tracer was developed. Acetylation of amine 3 with CH(3)COCl, followed by deprotection and aqueous workup, produced oseltamivir 4 from 3 within 10 min. The obtained 4 was sufficiently pure for PET studies. This method can be extended to PET tracer synthesis using CH(3)(11)COCl.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique

Synopsis Proteolytic enzymes, protease and trypsin have recently been introduced to reduce the inconsistency hitherto encountered in the unlabelled antibody-enzyme method using PAP. This study investigated factors determining the optimum conditions for use of such enzymes in order to establish which one is most suitable. Trypsin was the most effective enzyme; however, its activity decreased over 3 h, a feature paralleled immunocytochemically. Method and duration of fixation appears to influence the required time of exposure to trypsin in order that consistent immunostaining may be produced. Treatment of sections with trypsin prior to the use of the unlabelled antibody-enzyme method using PAP renders the technique reliable, provided the enzyme is used in a carefully controlled manner.