Indirect Immunoperoxidase Procedure Research Articles

Downregulation of the Cyclin-Dependent Kinase Inhibitor p27 kip1 Might Correlate with Poor Disease-Free and Overall Survival in Inflammatory Breast Cancer

Purpose The objective of this study was to evaluate whether p27 kip1 downregulation is a prognostic factor in patients with inflammatory breast carcinoma (IBC). Patients and Methods Fifty-eight patients with IBC were treated between January 1994 and July 2002. Median age was 49 years. Thirty-eight patients had baseline biopsy specimens. Patients received preoperative chemotherapy with FAC (5-fluorouracil/doxorubicin/cyclophosphamide; 34%) or FAC followed by a taxane (66%). All patients underwent mastectomies. All patients received radiation therapy and hormonal treatment when indicated. Expression level of p27 kip1 was evaluated by indirect immunoperoxidase procedure. The p27 kip1 was considered downregulated if nuclear staining was present in > 50% of the neoplastic cells. Results Thirty-two patients (84%) had p27 kip1-downregulated tumors, and 6 patients (17%) had p27 kip1-normal tumors. Six patients (16%) exhibited a pathologic complete response. At a median follow-up of 43 months, 25 recurrences (66%) and 27 deaths (71%) occurred. Patients with p27 kip1-downregulated tumors had fewer pathologic complete responses (9% vs. 50%; P = 0.03) and had lower 4-year recurrence-free survival (23% vs. 83%; P = 0.03) and overall survival rates (36% vs. 83%; P = 0.01). Conclusion The p27 kip1 deregulation manifested by low protein cellular concentration might represent an adverse prognostic marker in IBC and could provide a valuable tool for selecting treatment for this aggressive disease.

Transforming growth factor-beta isoform expression in the perisutural tissues of craniosynostotic rabbits.

To describe the expression patterns of the various transforming growth factor-beta (Tgf-beta) isoforms, known to be involved in suture development, in the perisutural tissues of rabbits with naturally occurring craniosynostosis and relate such differential expression to the pathogenesis of premature suture fusion. Twenty-one coronal sutures were harvested from six wild-type control New Zealand White rabbits and five rabbits with familial coronal suture synostosis at 25 days of age for histomorphometric and immunohistochemical analyses. Tgf-beta isoform immunoreactivity was assessed using indirect immunoperoxidase procedures with specific antibodies. Synostosed sutures had significantly (p <.01) greater bone area and relatively more osteoblasts and osteocytes in the osteogenic fronts, compared with wild-type sutures. Tgf-beta isoform immunoreactivity showed differential staining patterns between wild-type and synostosed perisutural tissues. In wild-type sutures, Tgf-beta1 and Tgf-beta3 immunoreactivity was significantly (p <.001) greater than Tgf-beta2 staining in all perisutural tissues. In synostosed sutures, the opposite pattern was observed, with Tgf-beta2 immunoreactivity significantly (p <.001) greater than Tgf-beta1 and Tgf-beta3 in the osteogenic fronts, dura mater, and periosteum. Findings from this study suggest that an overexpression of Tgf-beta2, either in isolation or in association with an underexpression of Tgf-beta1 and Tgf-beta3, may be related to premature suture fusion (craniosynostosis) in this pathological rabbit model. These abnormal expression patterns may be involved in premature suture fusion either through increased cell proliferation, decreased apoptosis of the osteoblasts or both at the osteogenic fronts.

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.

Immunoelectron microscopic study for histamine in the gastric enterochromaffin-like cells of rats treated with the proton pump inhibitor lansoprazole.

The enterochromaffin-like (ECL) cells of the gastric mucosa in animals play an important role in gastric acid secretion. They contain few granules and numerous secretory vesicles and microvesicles. They operate under the control of circulating gastrin. In the present study, we conducted an immunoelectron microscopic study for histamine (HA) in the ECL cells of rats given the proton pump inhibitor lansoprazole (LP), which is known to induce hypergastrinemia. The pre-embedding indirect immunoperoxidase procedure utilized a mouse monoclonal antibody AHA-2 against glutaraldehyde-conjugated HA. Rats received LP (50 microg/kg per day, subcutaneously) over a period of a month, and developed hypertrophy of the ECL cells in the stomach. It was clearly demonstrated that HA was located to a much higher degree in the cytoplasm of ECL cells of LP-treated rats than in normal rats. HA immunoreactivity was observed in the cores of the granules and secretory vesicles of the ECL cells in all the rats, but in the LP-treated rats it was observed in the cores of the newly developed vacuoles as well. These results may suggest that HA may be actively generated in the cytoplasm of the hypertrophic ECL cells of LP-treated rats. Also suggested in the present study is that HA is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement, and that vacuoles are formed by the fusion of large secretory vesicles. Furthermore, the finding that relatively little HA immunoreactivity existed in the vacuoles may suggest that the vacuoles actively degrade superfluous secretory products (for example, HA) through enhanced autophagocytosis and/or oxidative stress. Another possibility may be that the membrane-bounded structure regarded as the vacuoles in this study might actually be an invagination structure produced as a result of successive series of exocytosis through which the secretory vesicles actively and rapidly release HA.

Immunoelectron-microscopic demonstration of histamine depletion in the gastric enterochromaffin-like cells of rats treated with alpha-fluoromethylhistidine.

We conducted an immunoelectron-microscopic study for histamine (HA) in the enterochromaffin-like (ECL) cells of normal rats and rats given alpha-fluoromethylhistidine (alpha-FMH, 3 mg/kg per hour) via osmotic minipumps over a period of 24 h. The indirect immunoperoxidase procedure utilized a mouse monoclonal antibody (mAb), AHA-2, which is produced against glutaraldehyde-conjugated HA. alpha-FMH is a potent and irreversible inhibitor of the HA-forming enzyme histidine decarboxylase and is known to reduce tissue HA concentrations in several tissues. The present study clearly demonstrated that HA immunoreactivity, which was found to a high degree in the cores of the granules and secretory vesicles and in the cytoplasm of ECL cells of control rats, was completely abolished from the corresponding compartments in the cells of alpha-FMH-treated rats. Furthermore, treatment with alpha-FMH drastically lowered the number of secretory vesicles and was associated with larger cores in the granules of the ECL cells. These results seem to support the idea of a HA-pathway mechanism, emphasizing that the granules in normal ECL cells take up HA from the cytosol during its transport from the Golgi zone to the more peripheral portion of the cell and condense it in their cores, thus forming mature secretory vesicles. However, the present study showed that not only the secretory vesicles but also almost all the granules seen in ECL cells were already loaded with HA in their cores, suggesting that the newborn granules very rapidly take up HA from the cytosol. Also suggested was the fact that HA depletion impairs the maturation of the granules into secretory vesicles.

Expression of metallothioneins-I and -II isoforms at positive patch-test sites.

The expression and the distribution of metallothioneins (MT)-I and II isoforms were evaluated in 5 healthy volunteers and in 16 subjects with positive patch test reactions to various compounds. Skin specimens taken both from the healthy skin of the back and at positive patch test sites (at 48 h), were treated using a 3-step indirect immunoperoxidase procedure with a mouse monoclonal IgG1 antibody reactive against I and II isoforms of human, rat and horse MT. MT were expressed in the basal layer of the healthy skin of both controls and sensitive subjects, without any significant difference. At positive patch test sites, there was an overexpression of MT in basal and suprabasal layers of the epidermis. Overexpression of MT, related to the degree of the inflammatory reactions elicited by the penetrating compounds was observed in the dermis. The cells expressing MT in the dermis were mostly T lymphocytes and cells with dendritic morphology which positively stained in part for CD34 and in part for XIIIa markers and negatively for KP1, S100 and HLA-DR. Taken together, these results seem to indicate that MT represent a constitutive mechanism of defence expressed by different types of cells in the skin, which is triggered by contact with both metallic and non-metallic compounds. The biological significance of MT in the skin remains to be elucidated. Our preliminary findings do not permit evaluation of whether these nearly ubiquitous proteins exert their cytoprotective effects in the skin acting simultaneously as antioxidant, metal binding or zinc suppliers, or if they display these activities mainly depending on the nature of the penetrating substances.

Expression of CD44-v6-containing isoform on breast tissue adjacent to silicone breast implants.

Although a variety of studies exists, the silicone controversy is still going on. In the present study we investigated the expression of the activation markers CD44-v6, intracellular adhesion molecule 1 (ICAM-1), and HLA class II molecules on breast epithelia adjacent to a silicone gel-filled breast implants (SGBI). Using an indirect immunoperoxidase procedure 12 tissue samples from implant recipients were investigated and 6 tissue samples from patients with slight breast hyperplasia were used as controls. Expression of CD44-v6 was found on ductal epithelia in all specimens from implant recipients, HLA-DR was expressed in all specimens, HLA-DQ in 3 of 10, and ICAM-1 in 8 of 12. In contrast, neither CD44-v6 nor HLA-DQ nor ICAM-1 were expressed on ductal epithelia from patients with slight breast hyperplasia, and HLA-DR was only weakly expressed in 2 of 6 patients. These data show that CD44-v6 can be expressed by benign cells under certain conditions, which may indicate an abnormal state of activation in breast tissue adjacent to an SGBI.

Production and Characterization of Monoclonal Antibodies against Avian Reovirus Strain S1133

Monoclonal antibodies (MAbs) were produced against the avian reovirus strain S1133. MAbs were isotyped and used to develop diagnostic tests. Splenocytes from immunized mice were screened by enzyme-linked immunosorbent assay (ELISA). Two hybridomas secreted MAbs against avian reovirus S1133. One MAb secreted IgG1 and the other secreted IgG2a. All MAb light chains were kappa Specificity of MAbs was tested against four avian reovirus strains: S1133, 1733, CO8, and 2408. Strains S1133, 1733, and 2408 viruses were in the same subtype; the CO8 virus belonged to a different subtype. The MAbs reacted with all reovirus strains by ELISA, dot blot, immunofluorescence assay, and immunoblotting. No MAb had neutralizing activity against the tested reoviruses. Immunoblot analysis showed the one MAb bound to protein sigma A with molecular weight of 39,000 Daltons for all reovirus strains. Another MAb bound to the protein sigma C with an approximate molecular mass of 32,000 Daltons. An indirect immunoperoxidase (IP) procedure was developed using a MAb to detect reovirus in formalin-fixed, paraffin-embedded tissues from infected chickens and chicken embryo fibroblast cell cultures. The IP test was simple, fast, and economical and enabled simultaneous evaluation of viral antigen-producing cells with tissue pathologic changes confirming that the reovirus caused the lesions.

Identification and partial characterization of a unique marker for human basophils.

A unique marker for human basophils is needed to precisely determine the involvement of this cell type in clinical disease. To search for a marker of the basophil secretory granule, mouse hybridomas were generated against purified human basophils and screened for basophil-selective Ab. One hybridoma (2D7) produced an IgG1 kappa Ab that labeled basophils, but not lymphocytes, monocytes, eosinophils, neutrophils, and mast cells by an indirect immunoperoxidase procedure. The pattern of basophil staining was cytoplasmic and granular by light microscopy. By immunogold electron microscopy, the 2D7 ligand was localized to secretory granules. Activated basophils showed reduced 2D7-dependent staining intensity, consistent with a secretory granule localization. Tissue sections of normal skin, lung, and bowel showed no reactivity with 2D7, consistent with the anticipated absence of basophils in these tissues. 2D7 staining of basophils was clearly distinct from metachromatic staining, which was presumably dependent on proteoglycan. Extracts of normal human basophils subjected to Western blotting with 2D7 exhibited two predominant bands at apparent molecular masses of 76,150 and 72,260 Da. In summary, the 2D7 ligand appears to be a specific marker for human basophils and may facilitate the assessment of basophil involvement in diseases such as asthma, anaphylaxis, and atopic dermatitis.

Simultaneous immunoenzymatic staining of catecholamines, catecholamine-biosynthesizing enzymes, and bromodeoxyuridine in adrenal medullary cells of the rat.

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.

Presence of natural killer cells in human chronic periapical lesions.

Natural killer (NK) cells are lymphoid in nature and appear to kill target cells (neoplastic and virus-infected cells) without apparent prior immunization. Ten human chronic periapical lesions were examined for the presence of these cells. The lesions were collected, frozen in liquid nitrogen, and sectioned. They were then stained with an indirect immunoperoxidase procedure for the presence of human NK cells. Human tonsils and human periapical scar tissue were used as positive and negative controls. The results showed absence of NK cells in scar tissue and the presence of NK cells in tonsils and all samples of human periapical lesions. NK cells may play a defensive role in controlling root canal infections or, possibly, a role in its pathogenesis.

Use of an Immunoperoxidase Technique to Detect Equine Herpesvirus-1 Antigen in Formalin-Fixed Paraffin-Embedded Equine Fetal Tissues

An indirect immunoperoxidase (IP) procedure using the avidin-biotin-peroxidase complex detection technique was developed to detect viral equine herpesvirus-1 (EHV-1) antigen in formalin-fixed paraffin-embedded tissues from aborted equine fetuses. The procedure was applied to liver, lung, and other tissues from 20 cases of confirmed or suspected EHV-1-induced abortions. Specific staining was observed in tissue sections from EHV-1-infected fetuses. Positive IP staining was present in tissues of 7 cases that were also positive by fluorescent antibody (FA) and virus isolation (VI) and that had typical histologic lesions. There was no IP staining in 7 cases that had no histologic lesions and negative FA and VI results. Five cases had typical histologic lesions and positive results in only 1 laboratory test; 3 were positive by VI and 2 by FA. Liver of 1 case was positive by IP, but tissues were too autolytic for other tests to be evaluated.

Rapid diagnosis of infectious laryngotracheitis using a monoclonal antibody‐based immunoperoxidase procedure

An indirect immunoperoxidase (IP) procedure using monoclonal antibody was developed for detection of infectious laryngotracheitis (ILT) virus antigen in frozen tissue sections. This IP procedure was compared with an indirect immunofluorescent antibody (FA) procedure, histo-pathology and virus isolation for detection of ILT virus in tracheas of experimentally infected chickens. Compared with virus isolation, sensitivity and specificity of IP were 72 and 93%, respectively; sensitivity and specificity of FA were 53 and 90%, respectively. Histopathological detection of ILT virus infection was highly specific (98%), but sensitivity was poor (42%). These findings indicate potential usefulness of the IP procedure for ILT diagnosis.

Immunohistochemical localization of calbindins (28K and 9K) in the tissues of the baboon Papio ursinus

An indirect immunoperoxidase procedure was used to detect the presence of calbindin-D28K and calbindin-D9K in the cerebellum, kidney, and duodenum of the baboon Papio ursinus. Antibodies to chick calbinding-D28K and to both rat and mouse calbindin-D9K were used. The cerebellum and kidney were shown to contain calbindin-D28K; the doudenum contained calbindin-D9K. In the cerebellum, positive staining was found in the Purkinje cells only; in the kidney, positive staining was found in the distal convoluted tubules, connecting tubules, and collecting tubules, extending deep into the medullary regions of the kidney. Staining in the duodenum was confined to the enterocytes of the villi, with no stain present in the crypt regions or goblet cells. Thus the baboon, a primate, contains the larger of the calbindins in both the cerebellum and kidney as does the human and monkey, but its distribution in the kidney is more generalized than that found in humans. The molecular weight of calbindin-D9K was found to be similar to that found in other animals. However, the calbindin-D28K from the baboon tissues appears to be slightly smaller than the protein found in other animals and may therefore be of similar size to the human calbindin-D28K (Mr 26,000).

A Monoclonal Antibody-Based Immunoperoxidase Procedure for Rapid Detection of Infectious Bronchitis Virus in Infected Tissues

A monoclonal antibody (MAb)-based indirect immunoperoxidase (IP) procedure was applied to detect infectious bronchitis virus (IBV) antigen in frozen tissue sections. With this procedure, IBV antigen could be detected in chorioallantoic membranes (CAMs) within 15 hr postinoculation and in the respiratory tissues of chickens within 96 hr postinoculation. Endogenous peroxidases in chicken tissues were removed by treatment with periodic acid. The IP technique was highly sensitive, convenient, and economical. It allowed simultaneous evaluation of antigen-bearing cells and the general tissue morphology. The IP-stained slides also provided a permanent record.

Effects of transient forebrain ischemia on peptidergic neurons and astroglial cells: evidence for recovery of peptide immunoreactivities in neocortex and striatum but not hippocampal formation

The effects of transient (30') forebrain ischemia (4 vessel occlusion model) on peptidergic neurons and astroglial cells in various diencephalic and telencephalic areas have been analyzed. The study was performed at various time intervals of reperfusion, i.e. 4 h, 1, 7 and 40 days. Neuropeptide Y (NPY), somatostatin (SRIF), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP) and arginine-vasopressin (AVP) immunoreactive (IR) neuronal systems and glial fibrillary acidic protein (GFAP)-IR glial cells have been visualized by means of the indirect immunoperoxidase procedure using the avidin-biotin technique. The analysis was performed by means of computer assisted microdensitometry and manual cell counting. At the hippocampal level a huge reduction of neuropeptide (CCK, SRIF, VIP) IR cell bodies was observed, still present 40 days after reperfusion. On the contrary, in the frontoparietal cortex the number of the neuropeptide (CCK, SRIF, VIP, NPY) IR neurons showed a decrease at 4 h, 1 and 7 days after reperfusion followed by a complete recovery at 40 days. A rapid reduction followed by an almost complete recovery (7 days after reperfusion) was also observed at striatal level where SRIF- and NPY-IR neurons were detected. A marked decrease of NPY-IR terminals was observed in the paraventricular and periventricular hypothalamic nuclei and in the paraventricular thalamic nucleus. AVP-IR was markedly reduced in the magnocellular part of the paraventricular nucleus throughout the analyzed period (7 days after reperfusion). GFAP-IR was increased in the hippocampal formation and neostriatum while a not consistent increase was observed at neocortical level. These data point to a differential recovery of peptide-IR and to a different astroglial response in the various brain areas after transient forebrain ischemia. Region-specific factors rather than factors related to neuronal chemical coding seems to play a major role in determining the vulnerability of neuronal populations to transient ischemia.

A cytokeratin-immunohistochemical study of hepatoblastoma

Six cases of hepatoblastoma (five epithelial, one mixed epithelial-mesenchymal) were studied on serially cut cryostat sections, using a panel of monoclonal antibodies directed against individual cytokeratins, vimentin, and desmin, in an indirect immunoperoxidase procedure. Embryonic and fetal-type tumor cells expressed the “hepatocellular” cytokeratins no. 8 and 18 but, surprisingly, also expressed the “bile duct type” cytokeratin no. 19. In addition, two cases had a number of tumor cells which were also positive for the “bile duct type” cytokeratin no. 7. Cells embedded in osteoid-like material were immunoreactive for vimentin but also for cytokeratins no. 7, 18, and 19. Gel electrophoresis, and Western blotting of cytoskeletal extracts, confirmed the immunohistochemical data. The implications of these findings for the histogenesis of hepatoblastoma are discussed in this report.

Ureaplasma felinum sp. nov. and Ureaplasma cati sp. nov. Isolated from the Oral Cavities of Cats

Seven ureaplasma strains isolated from the oral cavities of domestic cats (Felis domestica) were characterized and compared with the type strains of the three previously established species of this genus, Ureaplasma urealyticum (humans), Ureaplasma diversum (cattle), and Ureaplasma gallorale (chickens). The feline strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked cell walls, passed through 0.45-micron membrane filters, required cholesterol for growth, and formed minute (15- to 140-microns) colonies on agar medium. The seven feline strains fell into two distinct groups based on (i) their antigenic properties (determined by using the metabolism and growth inhibition and indirect immunoperoxidase procedures), (ii) their genomic properties (determined by using DNA-DNA hybridization and DNA cleavage pattern procedures), and (iii) their polypeptide profiles (determined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses). Based on these properties, the two feline groups were unrelated to each other or to the three previously established species, and each group represents a distinct Ureaplasma species. Thus, we propose that ureaplasmas with these phylogenetic and genomic properties be given taxonomic status as Ureaplasma felinum and Ureaplasma cati, with strain FT2-B (= ATCC 49229 = NCTC 11709) and strain F2 (= ATCC 49228 = NCTC 11710) as the type strains, respectively.

Detection of Ehrlichia Risticii using an Avidin-Biotin-Immunoperoxidase Staining System

An indirect immunoperoxidase procedure using a specific anti-Ehrlichia risticii monoclonal antibody and an avidin-biotin-peroxidase staining method was used to detect E. risticii antigen in infected P388D1 murine monocytes. Several different methods of cytological fixation were used, including acetone (15 min), 95% ethanol (15 min), Bouin's fixative (5 hr), and 10% buffered neutral formalin (24 hr). The E. risticii organisms were labeled effectively and identified in cells fixed with acetone and ethanol. However, infected P388D1 cells fixed in 10% formalin or Bouin's fixative required enzymatic digestion with 1.0% trypsin for 15 min at 37 C before positive results were evident. This indirect immunoperoxidase avidin-biotin staining procedure proved to be a sensitive assay for the detection of intracellular E. risticii and may be an effective diagnostic procedure for formalin-fixed paraffin-embedded tissue.

Use of an Immunoperoxidase Test for the Detection of Bovine Herpesvirus-1 in Aborted Fetal Tissue

An indirect immunoperoxidase (IP) procedure using a specific monoclonal antibody and an avidin-biotin-peroxidase complex was developed and applied to detect virus antigen in formalin-fixed, paraffin-embedded tissue sections. This IP procedure was compared with currently used diagnostic tests for detection of virus-induced abortions caused by bovine herpesvirus-1 (BHV-1). The IP procedure was applied to detect BHV-1 antigen in sections of liver and lung from 87 aborted fetuses. Sixteen of these cases were positive for viral antigen by IP staining. Sections from both liver and lung were positive in 15 of the 16 cases. A fluorescent antibody test (FA), which was applied to acetone-fixed frozen sections of liver and lung, gave positive results on 12 of the 87 fetuses, 11 of which were also positive by IP. Seven of the 12 FA-positive cases were positive on both sections of liver and lung. When FA and IP were compared. FA had a sensitivity of 67% and IP had a sensitivity of 94%. Virus was isolated from one of the 67 cases tested. The tissues in which antigen was most frequently detected by IP were liver, lung, and kidney. Distinct multifocal staining was seen in positive sections of all these tissues.