Double Immunoenzymatic Staining Research Articles

The Y2 receptor agonist PYY3–36 increases the behavioural response to novelty and acute dopaminergic drug challenge in mice

The gastrointestinal hormone PYY(3-36) is a preferential Y2 neuropeptide Y (NPY) receptor agonist. Recent evidence indicates that PYY(3-36) acts on central dopaminergic pathways, but its influence on dopamine-dependent behaviours remains largely unknown. We therefore explored the effects of peripheral PYY(3-36) treatment on the behavioural responses to novelty and to dopamine-activating drugs in mice. In addition, we examined whether PYY(3-36) administration may activate distinct dopamine and γ-aminobutyric acid (GABA) cell populations in the mesoaccumbal and nigrostriatal pathways. We found that i.p. PYY(3-36) injection led to a dose-dependent increase in novel object exploration. The effective dose of PYY(3-36) (1 μg/100 g body weight) also potentiated the locomotor reaction to the indirect dopamine receptor agonist amphetamine and increased stereotyped climbing/leaning responses following administration of the direct dopamine receptor agonist apomorphine. PYY(3-36) administration did not affect activity of midbrain dopaminergic cells as evaluated by double immuno-enzyme staining of the neuronal early gene product c-Fos with tyrosine hydroxylase. PYY(3-36) did, however, lead to a marked increase in the number of cells co-expressing c-Fos with glutamic acid decarboxylase in the nucleus accumbens and caudate putamen, indicating activation of GABAergic cells in dorsal and ventral striatal areas. Our results support the hypothesis that acute administration of the preferential Y2 receptor agonist PYY(3-36) modulates dopamine-dependent behaviours. These effects do not seem to involve direct activation of midbrain dopamine cells but instead are associated with neuronal activation in the major input areas of the mesoaccumbal and nigrostriatal pathways.

Labeling of Multiple Cell Markers and mRNA Using Automated Apparatus

Double immunoenzymatic labeling of 2 different molecules in tissue sections is a widely used technique. However, it is time consuming since the 2 immunoenzymatic procedures are carried out in sequence, and they must also be optimally performed to avoid unwanted background labeling. In this paper, we report that double immunoenzymatic staining performed using automated immunostaining apparatus considerably reduces the requirements in terms of time and is also highly reproducible and free of background. Three tissue markers can also be visualized by performing (after immunoperoxidase labeling) 2 sequential immuno-alkaline phosphatase procedures using different substrates. Furthermore, single or double detection of mRNA by in situ hybridization can be combined with immunoenzymatic labeling. Finally, automated labeling could also be performed on peripheral blood and bone marrow smears, opening the possibility of using this procedure in the analysis of hematologic/cytology samples.

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse, and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.

The FOXP1 Transcription Factor is Expressed in the Majority of Follicular Lymphomas but is Rarely Expressed in Classical and Lymphocyte Predominant Hodgkin’s Lymphoma

The expression of the FOXP1 forkhead/winged helix transcription factor has been investigated in normal and neoplastic lymphoid tissues using the FOXP1-specific monoclonal antibody, JC12. Using single and double immunoenzymatic staining, FOXP1 expression has been studied in a series of classical and lymphocyte predominant Hodgkin's lymphomas, follicle centre lymphomas and Hodgkin's lymphoma-derived cell lines. Neoplastic cells in the majority of classical and lymphocyte predominant Hodgkin's lymphoma were FOXP1-negative. In two cases of classical Hodgkin's lymphoma, the tumour cells showed mislocalisation of FOXP1 to the cytoplasm, while in one case of lymphocyte predominant Hodgkin's lymphoma, and in the Hodgkin's lymphoma cell line KMH2, scattered tumour cells showed nuclear expression of FOXP1. In contrast, the tumour cells in the majority of follicle centre lymphomas showed strong nuclear FOXP1 reactivity. Double labelling studies of lymphoid tissue indicated that a variable proportion of CD20-positive germinal centre B cells express FOXP1 while the vast majority of CD30-positive cells are FOXP1-negative. The heterogeneity of FOXP1 expression in germinal centre-derived lymphomas, may have more to do with the transforming events underlying these distinct types of lymphoma than with their common origin.

An antibody raised against in vitro-derived human mast cells identifies mature mast cells and a population of cells that are Fc epsilon RI(+), tryptase(-), and chymase(-) in a variety of human tissues.

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immunolabeling with 2C7 and tryptase, chymase, Fc epsilon RIalpha, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase(+) or chymase(+) mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc epsilon RIalpha. However, there were a significant number of Fc epsilon RIalpha(1) cells in all tissues studied that were tryptase(-) and/or chymase(-). In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93-96% of the Fc epsilon RIalpha(1) cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase(+) or chymase(+) cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase(+) cells in colon and stomach were c-kit(+). These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc epsilon RIalpha(1), chymase(-), and tryptase(-) cells in a variety of human tissues.

Immunohistochemical detection of interferon-gamma: fake or fact?

Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNgamma immunohistochemistry on tissue sections with a large panel of anti-IFNgamma antibodies. Thirteen different commercially available anti-IFNgamma antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin-biotin-peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNgamma-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. In vitro phorbol myristate acetate/ionomycin-stimulated T-cells served as positive control; unstimulated cells served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNgamma antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNgamma immunohistochemical single- and double staining analysis of the tissue sections showed huge variations in staining patterns, including positivity for smooth muscle cells (n = 8), endothelial cells (n = 4), extracellular matrix (n = 4), and CD138+ plasma cells (n = 12). Specific staining of T-cells, as the sole positive staining, was not achieved with any of the 13 antibodies. IFNgamma-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNgamma antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNgamma-immunohistochemistry must be interpreted with great caution.

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.

Morphometric analysis of pediatric and nonhyperplastic prostate glands: evidence that BPH is not a unique stromal process.

Although quantitative morphometry of benign prostatic hyperplasia (BPH) has been described, there is a paucity of information on the morphometry of the nonhyperplastic prostate. This study determines the histologic composition of prostates obtained from males, ages 2 days to 40 years, in order to provide insights into the morphometry of the "normal" gland. The histologic composition of 45 prostates was obtained from autopsies of males with age groups stratified to reflect the neonatal, childhood, peripubertal, adolescent, and young adult periods. Double immunoenzymatic staining and computer image analysis were used to determine the mean area densities of the smooth muscle (SM), connective tissue (CT), glandular epithelium (E), and lumen (L). A progressive decrease in SM area density throughout childhood, prepuberty, and puberty was seen. The density of SM significantly increased following puberty and throughout adolescence and early adulthood. There was a concomitant increase in CT from the neonatal period throughout childhood, prepuberty, and puberty, and a decrease after puberty and throughout adolescence and early adulthood. Since the changes in SM and CT were inversely related, the percent contribution of the stromal compartment to the total gland remained constant. The stromal to epithelial ratio remains constant from birth to age 40 in nonhyperplastic glands and is similar to the ratios in asymptomatic and symptomatic BPH tissues.

Merkel cells participate in the induction and alignment of epidermal ends of arrector pili muscles of human fetal skin

The follicular proximal ends of arrector pili muscles anchor the bulge areas of human hair follicles. However, the location of the ends of these muscles towards the epidermis, especially the fine points of attachment, is not fully understood. Conventional serial sections of the scalp skin of a 12-month-old infant were examined. Most of the distal ends were situated in the upper dermis, while some seemed to be in close contact with the epidermal basal layers. In vertical sections of the scalp skin of 36-week-old fetus, double immunoenzyme staining showed a close topographic correlation between the epidermal ends of the arrector pili muscles and the epidermal and dermal Merkel cells. These findings allow speculation that fetal Merkel cells participate in the induction and alignment of arrector pili muscles.

Comparison of the cellular composition of benign prostatic hyperplasia in chinese and caucasian-american men

Objectives To compare the cellular composition of benign prostatic hyperplasia (BPH) in Chinese and Caucasian-American men. Methods Surgical specimens of the prostate were obtained from 9 Chinese and 8 Caucasian-American men undergoing cystoprostatectomy for invasive transitional cell carcinoma. The mean ages of the Chinese and Caucasian-American men were 66.8 years and 66.4 years, respectively ( P = 0.94). The mean prostate weight of the Chinese and Caucasian-American men was 53.4 g and 32.1 g, respectively ( P = 0.01). Double immunoenzymatic staining with antibodies against actin and prostatic acid phosphatase and computer-assisted color image analysis were performed on whole-mount tissue sections. The percent area density of smooth muscle (SM), connective tissue (CT), epithelium (E), and epithelial lumen (L) were obtained by analyzing 30 fields from each specimen. Results The mean percent area density of SM, CT, E, and L in the prostate of Chinese men was 32%, 9.1 %, 10.8%, and 48.5%, respectively. The mean percent area density of SM, CT, E, and L in the prostate of Caucasian-American men was 52.5%, 27.9%, 12.8%, and 7%, respectively. Overall, the prostates of Chinese men contained significantly more glandular lumen and significantly less SM and CT. Conclusions The present study demonstrates that the cellular composition of BPH in the prostates of Caucasian-American and Chinese men is different. These cellular differences may account for previously observed differences in the incidence of clinical BPH.

A Rapid Immunoenzyme Double Labeling Technique Using EPOS Reagents

We present a rapid method for a double immunoenzyme staining of formalin fixed, paraffin embedded tissue sections using antibodies labeled with a peroxidase enzyme polymer system (EPOS) recently introduced by Dako. This method is rapid, taking less than 3 hr, is virtually background free, and has a sensitivity comparable to the more cumbersome indirect methods. (The J Histotechnol 18:35, 1995)

Double epi-illumination microscopy with separate visualization of two antigens: a combination of epi-polarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining.

We present a method for an epi-illumination immunohistochemical double staining approach. The method combines the use of an immuno-alkaline phosphatase technique and the immunogold-silver technique, visualized with epifluorescence and epi-polarization illumination, respectively. Out of six tested alkaline phosphatase activity-revealing methods, only the reaction product obtained with the Becton Dickinson CAS Red kit showed an intense red fluorescence with a rhodamine filter set and no signal with epi-polarization illumination. The silver precipitate did not exhibit any signal with the rhodamine filter set. This allows separate observation and photographic recording of two antigens in one tissue section, an objective that cannot be achieved with conventional immunoenzyme double staining methods. The double epi-illumination approach presented is compatible with different immunoenzyme double staining protocols.

Multiparameter analysis of primary epithelial cultures grown on cyclopore membranes.

The use of porous membranes as culture support for epithelial cells has previously been shown to cause functional differentiation of these cells mimicking an in vivo condition, in contrast to culture on plastic. The different materials of which the membranes are made also have different properties, such as transparency, rigidity, and retention of molecules. Cyclopore membranes (polyethylene terephtalate) are permeable, transparent, rigid, and have low protein retention. In this study we examined the applicability of assessing multiple parameters on a single culture of primary epithelial cells on a Cyclopore membrane. Cultures of transitional epithelial cells on these membranes differentiate into an organoid-like epithelium. We were able to perform morphometric analysis during and after cell culture and to quantitate proliferation and differentiation by double immunoenzymatic staining. On these cultures, quantitative radiochemical analysis could also be achieved, retaining the morphology and the immunohistochemical staining. Cross-sections of paraffin-embedded and plastic-embedded cultures were analyzed qualitatively by light and transmission electron microscopy, respectively. Finally, cytokeratins in these cultures could also be visualized by immunofluorescence analysis. This suitability for simultaneous assessment of both qualitative and quantitative parameters on a single cell culture grown on a Cyclopore membrane reduces the need of biological materials and may lead to better insight into physiological processes.

The cell blot assay in analysis of rat anterior pituitary cell secretion

We have investigated the efficacy of the cell blot assay in analysis of the secretion of hormones and peptides from rat anterior pituitary cells. The dissociated cells are cultured on pieces of translucent polyvinylidene difluoride membrane, on which their secretory products are adsorbed and subsequently immunostained. The area and integrated optical density of the stained 'halo' surrounding individual cells is measured by microscopical image processing and the values for basal secretion of a particular hormone or peptide are compared with those after application of secretagogues or inhibitors. Our experiments tested established responses of dissociated rat anterior pituitary cells; in general, the results were as expected. Double immunoenzymatic staining could be used to show secretion of two products from the same or different cells in one preparation, and immunofluorescence with fluorescein- and/or rhodamine-labelled antibodies could be used instead of enzyme-linked immunolabelling. Optimal dilutions of immunoreagents were much higher than those used for immunocytochemistry on tissue sections. Although the cell blot assay does not provide absolute quantification, since some of the secreted product escapes into the medium, it is a relatively easy and economical way for morphologists to compare secretion from individual cells under varying conditions.

平行反応による酵素抗体二重染色法 異なるｉｓｏｔｙｐｅのモノクローナル抗体を用いて

平行反応による酵素抗体二重染色法 異なるｉｓｏｔｙｐｅのモノクローナル抗体を用いて

Characteristics of cloned cells of mixed müllerian tumor of the human uterus carcinoma cells showing myogenic differentiation in vitro

To elucidate the relationship between the epithelial and mesenchymal elements of malignant mixed Müllerian tumors (MMMT), the authors examined the biologic properties of two clones of different cell types (designated as FU-MMT-2-C1 and FU-MMT-2-S1) established from a uterine MMMT cell line (FU-MMT-2), which they previously have reported. By morphologic and immunocytochemical analyses, FU-MMT-2-C1 exhibited features of adenocarcinoma cells, whereas FU-MMT-2-S1 showed characteristics of sarcoma cells with myogenic differentiation. Some FU-MMT-2-C1 cells at confluence differentiated spontaneously into myogenic mesenchymal cells in vitro. In addition, transitional-type cells between epithelial cells and sarcomatous cells were observed in the areas of mesenchymal differentiation in FU-MMT-2-C1 by light and electron microscopic study. Ultrastructurally, the transitional-type cells represented biphasic morphologic characteristics consisting of epithelial and myogenic features, which proved to coexpress epithelial, mesenchymal, and muscle markers by double immunoenzymatic staining. However, no epithelial differentiations were apparent in the sarcoma clone FU-MMT-2-S1. FU-MMT-2-C1 produced tumor in nude mice, the histologic study of which showed a mixture of adenocarcinoma and myogenic sarcomatous elements that resembled the original tumor. Cytogenetic studies demonstrated that these two clones were monoclonal in origin because of the presence of common karyotypic abnormalities in both cells. In addition, the amplified (approximately fourfold to eightfold) c-myc oncogene was found in the cloned cells and the original tumor cells. The current results strongly support the theory of single cell origin in uterine MMMT and suggest that the mesenchymal elements originated from primitive Müllerian epithelial cells capable of differentiating into epithelial, mesenchymal, or both elements.

Practical suggestions for successful immunoenzyme double-staining experiments.

Many methodologies exist to perform an immunoenzyme double staining. Hence, the practical problem arises as to which of these methods is optimal for one's own experimental design. A process of selection is described which is derived from our own practical experience. First, a general strategy is outlined for the handling of tissue sections to be used for multiple staining methods. Secondly, the selection of an appropriate immunoenzyme double-staining concept is made using a flow chart. Thereafter we give criteria for the definitive selection of an immunoenzyme double-staining protocol based on the characteristics of the tissue or cell type under study. Particular attention is given to the selection of appropriate detection systems, applying enzymes or gold particles, and good contrasting colour combinations. The problems of visualizing co-localization using immunoenzyme double staining are dealt with, and suggestions are made to adapt the method, if necessary, in order to optimize it.

Quantitative Morphometry of the Adult Human Bladder

The primary objective of the present retrospective study was to characterize the effects of aging and BPH on bladder morphometry. Eighty-six bladder specimens were obtained from the autopsy archives of the Milwaukee County Medical Complex. The bladder specimens were divided into 4 groups based upon age and gender: Group I: males between the ages of 35–45 years; Group II: males between the ages of 65–75 years; Group III: females between the ages of 35–45 years; and Group IV: females between the ages of 65–75 years. The age groups were selected in order to identify a group of males with and without BPH. The area density of smooth muscle:connective tissue was determined in bladder specimens using color assisted computer image analysis. Masson-trichrome and double immunoenzymatic staining techniques were used to discriminate the smooth muscle and connective tissue elements of the bladder. The area density of smooth musclexonnective tissue in the Masson-trichrome stained sections was significantly greater in Group I vs. Group II (2.90 ± 0.22 vs. 2.33 ± 0.16) and in Group III vs. Group IV (2.85 ± 0.13 vs. 2.03 ± 0.20). Aging was associated with a decrease in the area density of smooth musclexonnective tissue ratio in both males and females. The area density of smooth musclexonnective tissue was not significantly different in younger males and females (Group I vs. Group III) and older males and females (Group II vs. Group IV). The present morphometric study suggests that aging and not BPH, is associated with a relative increase in detrusor fibrosis. Infravesical obstruction in BPH may effect bladder function via mechanisms unrelated to the histologic composition of the bladder. Accepted for publication March 12, 1992.

Association of the mycobacterial 30-kDa region proteins with the cutaneous infiltrates of leprosy lesions. Evidence for the involvement of the major mycobacterial secreted proteins in the local immune response of leprosy.

The granulomatous skin lesions of human leprosy are known to be due to the cutaneous immune reaction to various mycobacterial antigens. In the present study, by immunohistochemical analysis using a previously characterized monoclonal antibody (MoAb) 3A8 we have demonstrated a selective expression of the 3A8 epitope of mycobacterial 30-kDa proteins, the major secreted proteins of mycobacteria, in various forms of leprosy lesions across the clinical spectrum. The localization of MoAb 3A8 staining is confined to the areas of cellular infiltrates of the lesions. In tuberculoid lesions the intense 3A8 staining was seen mostly in association with the membrane of the dermal cellular infiltrates whereas in highly bacilliferous lepromatous lesions the staining seems to be diffused with granular appearance but not in the form of bacteria. In patients with reversal reaction the staining was specifically extended to cells infiltrating the epidermis. MoAb 3A8 did not show any reactivity with inflammatory skin lesions of patients other than those with leprosy. Since the 3A8 epitope of 30-kDa proteins has been shown to be present in all cellular compartments of the mycobacteria and in the actively secreted BCG 85 antigen complex, MoAb 3A8 reactive protein(s) in leprosy lesions may be derived either from degraded somatic mycobacterial products or from antigens actively secreted by live bacilli. The latter could be true in the cases of untreated lepromatous lesions with high bacterial load since live M. leprae has also been considered to secrete corresponding 30-kDa proteins similar to other closely related mycobacteria. By double immunoenzyme staining we clearly demonstrate the expression of 3A8 epitope on CD68+ macrophages in the granulomas of tuberculoid leprosy, whereas in highly bacilliferous lepromatous lesions most of the double staining was seen in a diffuse pattern within the interstitial space of the cellular infiltrate as well as in the cytoplasm of CD68+ macrophages. In lesions from reversal reaction the 3A8 epitope is more strongly expressed on CDla+ dendritic Langerhans cells (LC) both in the epidermis and in the dermis as compared with other types of leprosy. This provides evidence for the involvement of LC in handling of mycobacterial antigenic epitopes in leprosy lesions. Further, immunoenzyme double staining revealed that the expression of this mycobacterial 3A8 epitope on antigen presenting cells such as CD68+ macrophages and CDla+ LC is present in juxtaposition with CD3+ T cells including the alpha beta and gamma delta receptor-bearing T cells in the granuloma.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantifying the Smooth Muscle Content of the Prostate Using Double-Immunoenzymatic Staining and Color Assisted Image Analysis

The primary objective of the present study was to develop a method for quantifying the smooth muscle content of the prostate adenoma. A double immunoenzymatic staining technique was coupled with color assisted image analysis to determine the area density of the smooth muscle within the prostate adenoma. Eight males with symptomatic BPH underwent transrectal biopsy of the prostate. Four micron thick tissue sections were used for the double immunoenzymatic staining process. Rabbit anti-desmin and mouse anti-human prostatic acid phosphatase antibodies were used to selectively bind smooth muscle and prostatic epithelium, respectively. The two different tissue antigens were identified with peroxidase-antiperoxidase (PAP) and alkaline phosphatase-antialka-line phosphatase techniques. The alkaline phosphatase activity and peroxidase activity were developed with fast red and DAB chromogens. The BQ MEG IV Vista color system image analysis was used to discriminate color differences from the stained tissue sections. The thresholds were set to identify smooth muscle (dark brown), epithelium (red), fibrous tissue (pale brown), and glandular lumina (colorless). The mean area density of smooth muscle, fibrous tissue, glandular epithelium, and glandular lumina was 22%, 54%, 16%, and 9%, respectively. The present study suggests that a significant component of the prostate adenoma is smooth muscle. The application of this technique will be utilized to provide further insights into the role of smooth muscle in the pathogenesis and therapy of BPH.