Journal of Histochemistry & Cytochemistry

A method was developed for detection of bromodeoxyuridine (BrdU) in conjunction with other antigens in formalin-fixed paraffin sections with microwave antigen retrieval. The method was applied to rat adrenal medulla to demonstrate S-phase nuclei in epinephrine-producing cells stained for immunoreactive phenylethanolamine-N-methyltransferase and in norepinephrine-producing cells stained for immunoreactive tyrosine hydroxylase. The quality of staining for all three antigens was comparable to or better than that previously obtained with other techniques. This method provides an efficient tool for studying turnover of subpopulations of adrenal chromaffin cells. It should also be widely applicable to other cells and tissues.

We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-embedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from barely to highly discernible and interpretable, with virtually no nonspecific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHrP mRNA expression was confined to the basal and spinous keratinocyte layers of skin. There was strong hybridization in the spinous keratinocyte layer and a low level of hybridization in the basal layer. An extensive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcinomas and basal cell carcinomas of the skin, from pathology archives, were examined for the presence of PTHrP mRNA. The results reflected previous immunohistochemical studies, with every squamous cell carcinoma hybridizing strongly with the PTHrP probes. The basal cell carcinomas showed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynecological tract also reflected the immunohistochemical findings, with expression found in the squamous cell carcinomas but not in the adenocarcinomas. In situ hybridization with digoxigenin-labeled oligonucleotide probes and antibody enhancement has provided a sensitive, highly specific procedure for detection of PTHrP mRNA in tumors and normal tissue.

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

We designed an improved method to observe proliferating cells with well-preserved ultrastructure. After IP injection of bromodeoxyuridine (BrdU) into rats, the pituitaries were fixed in 4% paraformaldehyde with 0.05% or 0.2% glutaraldehyde and post-fixed with ferrocyanide-reduced osmium. They were embedded in LR White and polymerized by heat. BrdU incorporated into DNA was detected with a commercial anti-BrdU monoclonal antibody (MAb) by the immunogold or the immunogold-silver staining method. Using these methods, proliferating cells labeled by BrdU were observed with well-preserved ultrastructure. By light microscopy, the number of labeled cells was almost the same regardless of the fixative used. By electron microscopy, localization of gold particles that indicate incorporated BrdU varied according to the cells and was mainly observed in two patterns, one in which gold particles were localized in condensed chromatin scattered in the nucleus and the other in which gold particles were dispersed evenly all over the nucleus. These results showed that with our improved method fine ultrastructure and good immunoreactivity of BrdU can be obtained in proliferating cells. We consider that this method is very useful for ultrastructural study of cell proliferation and differentiation.

Since its introduction, the avidin-biotin-peroxidase (ABC) complex has become an invaluable detection system for a wide variety of bioanalytical applications. In these techniques, avidin and biotin-peroxidase are mixed at a pre-determined ratio so that the soluble ABC complex retains biotin binding sites. Consequently, the complex contains an excess of avidin over biotinylated peroxidase residues. On theoretical considerations, however, an ABC complex designed for maximal signal intensity must consist of an excess of peroxidase over avidin molecules. Therefore, ABC complexes with reversed molar ratios of biotinylated peroxidase to avidin (rABC complexes) were prepared and an intermediate streptavidin step was introduced to bind the rABC complexes to biotinylated IgG molecules. The signal generating power of this new streptavidin-rABC sequence proved superior to that of the conventional ABC technique in ELISA assays and in immunostaining of tissue sections.

The rat adrenal medulla consists mainly of low proliferating, highly differentiated parenchymal cells. By immunocytochemical techniques, two types of parenchymal cells can be identified, norepinephrine (NE)- and epinephrine (E)-storing cells. Bromodeoxyuridine (BrdU), a thymidine analogue often used to identify proliferating cells, can also be detected by immunocytochemical techniques. We developed double- and triple-labeling procedure(s) for simultaneous visualization of NE, E, dopamine beta-hydroxylase (DBH), phenylethanolamine-N methyltransferase (PNMT), and BrdU in rat adrenal medulla. BrdU was administered to 7-week-old Wistar rats by mini-osmotic pumps. Tissues were fixed by perfusion with 4% paraformaldehyde and embedded in paraffin. By immunocytochemistry, first NE, E, DBH, and/or PNMT was detected by an indirect immunoalkaline phosphatase technique with Fast Red or Fast Blue as substrate. Next, incorporation of BrdU was detected with an indirect immunoperoxidase procedure using diaminobenzidine (DAB). Both NE- and E-storing cells, as well as endothelial cells, can incorporate BrdU, i.e., are able to divide. Occasionally, we also found BrdU-stained mitotic figures in E, PNMT and DBH immunoreactive cells. No BrdU incorporation was found in the post-ganglionic neurons of the adrenal medulla. The procedures described enable a detailed cell kinetic study of the NE- and E-storing cells in the adrenal medulla, particularly in the rat, which can lead to a better understanding of cell renewal in the adrenal medullary tissue under normal and pathological conditions.

The human MxA protein can be detected in the cytoplasm of IFN-alpha/beta-treated cells, whereas other cytokines, including IFN-gamma, are poor inducers. Because IFN-alpha/beta is predominantly synthesized in response to viral infections, MxA protein should be detectable in virally infected tissue. Biopsy specimens (n = 64) of 12 different dermatoses were therefore screened with an MxA-specific monoclonal antibody on formalin-fixed, paraffin-embedded and microwave-treated tissue sections. As expected, high amounts of MxA protein were found in acute viral skin lesions (chickenpox, Herpes zoster, and Herpes labialis). In addition, MxA protein was also detected in some inflammatory skin lesions of unknown etiology (lupus erythematosus, lichen planus, Schoenlein-Hennoch's anaphylactoid purpura and psoriasis). MxA protein was not found in non-viral infections (bacterial, mycotic, and parasitic) and was also not detectable in various other dermatoses (eczema, scleroderma, urticaria, granulomatous and bullous disorders). MxA staining proved a reliable, sensitive histochemical viral marker for infectious dermatoses. The positive results in non-infectious inflammatory dermatoses might implicate viral involvement or activation of the IFN system by thus far unknown mechanisms.

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

The rat kallikrein family consists of multiple closely related proteins. Most of these proteins have been purified from rat submandibular gland (SMG), among them rK9, an enzyme that has a direct vasoconstrictory effect on isolated aortic rings. Recently, two members of the kallikrein family, rK8 and rK9, were also purified from rat prostate, whereas other family members were not detected in this organ. In the present study, the immunohistochemical localization of rK9 was determined in rat prostate by the indirect immunofluorescence technique with polyclonal antisera against rat SMG rK9 in the first layer. The crossreactivity pattern of the antiserum against rat prostate tissue extract was established by flat-bed isoelectrofocusing and immunoblotting. The antiserum reacted with prostate rK9 but not with prostate rK8. Specificity of the immunofluorescent staining reaction was verified by staining with the primary antiserum after addition of purified SMG or prostate rK9, or other members of the kallikrein family including prostate rK8. rK9-specific immunofluorescence was detected in the prostate ductal structures. In the distal (acinar) segments, rK9 was found in the cytoplasm as granular staining in the middle third of the cell, in the luminal cell wall, and in the secretion within the lumen. In the intermediate segments, strong rK9-specific staining was observed adhering to the luminal wall as well as abundantly within the ductal lumen.

We used immunocytochemistry to detect the 90 KD major heat shock protein (HSP90), a potential regulator of gene expression, during male and female rat gonad development. In the Day 13.0 post-coital (dpc) fetal gonad, strong immunoreactivity to anti-HSP90 antibody was shown in the cytoplasm of primordial germ cells (PGCs). Other somatic cells in the gonad showed only faint reactivity. During testicular development, strong immunostaining was observed in the cytoplasm of embryonic germ cells and in spermatogonia and spermatocytes of the pre-pubertal testis. In adult testis reactivity of spermatogonia and pachytene spermatocytes was strong but reactivity of post-meiotic spermatogenic cells, i.e., secondary spermatocytes and spermatids, was extremely reduced. During ovarian development, immunostaining was also observed in the oogonia and the oocytes of pre-pubertal ovary. However, the staining of oocytes was reduced with the development of primordial follicles during the first week after birth. This study revealed that HSP90 is highly expressed in PGCs and continues to be expressed in both male and female pre-meiotic germ cells. The HSP90 accumulation may be essential for both male and female mammalian pre-meiotic germ cells.

We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.

Vitamin K-dependent protein S is an anticoagulant plasma protein that functions as a co-factor to activated protein C in the degradation of coagulation factors Va and VIIIa. We investigated the tissue/cellular distribution of protein S synthesis by Northern blotting, in situ hybridization, and immunohistochemistry. Northern blotting together with in situ hybridization, using specific oligodeoxynucleotide probes, demonstrated protein S mRNA in liver, lung, testis, epididymis, ovary, uterus, and brain. In the reproductive system, protein S mRNA was present in the cytoplasm of Leydig cells, interstitial cells of the ovary, epithelial cells of the epididymis, and in the endometrium, including endometrial mucous glandular membrane in the myometrium. Bronchial epithelial cells and alveolar macrophages were positive in the respiratory system. In the central nervous system, pyramidal neurons in the cerebral cortex and in the hippocampal region, and dentate fascia neurons gave strongly positive signals. Immunohistochemistry with monoclonal antibodies yielded a staining pattern that correlated well with results of in situ hybridization. In conclusion, results from Northern blotting, in situ hybridization, and immunohistochemistry suggested that rabbit protein S is expressed in several extrahepatic tissues. The presence of protein S transcripts in these fully differentiated cells suggests a cell type-specific gene expression which may be related to local anticoagulation or to other as yet unknown protein S functions.

We report a simple and reliable method for detection of two or more antigens within tissue sections by indirect immunoenzyme staining using mouse monoclonal antibodies (MAbs). This technique involves treating sections with two 5-min microwave (MW) oven heatings between sequential rounds of three-layer immunoenzyme staining (mouse MAb, goat anti-mouse IgG, and mouse PAP or mouse APAAP) and color development. Discrete staining of cell surface, cytoplasmic, and nuclear antigens was evident within individual cells. This technique has a number of advantages over those currently available. First, MW treatment denatures bound antibody molecules, thereby completely blocking crossreactivity between sequential rounds of staining. This allows the use of primary (and other) antibodies raised in the same species and the use of a sensitive three-layer staining method. Second, antigen retrieval after MW treatment markedly increases the sensitivity of cytoplasmic and nuclear antigen detection. Third, inactivation of peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes prevents inappropriate color development. Finally, this method can be used in both paraformaldehyde-fixed cryostat sections and formalin-fixed paraffin tissue sections. In conclusion, this is a simple, reliable, and sensitive technique that will be useful in many areas of diagnosis and research.

We describe a method for observing three-color immunofluorescence simultaneously in a histological section under a confocal laser scanning microscope. In this study we investigated the spatial relationship of blood vessels, reactive microglia, and amyloid beta-protein (A beta) deposits in post-mortem brain tissue of patients with Alzheimer's disease. HLA-DR was employed as a marker for activated microglia and von Willebrand factor (vWF) as a marker for vascular endothelial cells. HLA-DR was labeled with R-phycoerythrin (R-PE) and vWF with fluorescein isothiocyanate. A beta-protein was immunostained with the tandem conjugate of R-PE and cyanin 5. Three images were obtained serially by scanning a tissue section with a 488-nm laser beam in combination with an appropriate emission filter for each fluorochrome. Overlaying of these three images permitted simultaneous observation of three distinct structures: activated microglia, blood vessels, and A beta deposits. This technique provides an improved way to study the localization of three different antigens in a single tissue section.