Manual hematoxylin and eosin staining of mouse tissue sections.

The hematoxylin and eosin (H&E) stain is the standard used for microscopic examination of tissues that have been fixed, processed, embedded, and sectioned. It can be performed manually or by automation. For economic reasons, the manual technique is generally the method of choice for facilities with a low sample volume. This protocol describes manual H&E staining of fixed, processed, paraffin-embedded, and sectioned mouse tissues. In H&E-stained tissues, the nucleic acids stain dark blue and the proteins stain red to pink or orange. For accurate phenotyping and delineation of tissue detail, the protocol must be adhered to rigorously. This includes frequent reagent changes as well as the use of "in-date" reagents. Appropriate color in a good H&E stain allows for identification of many tissue subtleties that are necessary for accurate diagnosis.

Manual immunohistochemistry staining of mouse tissues using the avidin-biotin complex (ABC) technique.

There are many variations on the immunohistochemistry (IHC) procedure, but all are based on attachment of a primary antibody to a unique epitope on or within the cell. This step is followed by incubation of the cell/primary antibody complex with another, secondary antibody that recognizes the species in which the primary antibody was produced. The secondary antibody has an indicator molecule attached to it. The indicator produces a colored reaction product at the site of original epitope, allowing visualization. This basic two-antibody "sandwich" procedure has many modifications that include other layers of antibodies and numerous indicators, but all variations depend upon the unique ability of antibodies to recognize specific epitopes or antigenic determinants. The procedure described here is called the ABC (avidin-biotin complex) technique. The method utilizes the high avidity of biotin for avidin, which allows formation of a strong bond. The reagents described in this technique produce a gold/brown reaction product that identifies the epitope of interest.

Analysis of mouse model pathology: a primer for studying the anatomic pathology of genetically engineered mice.

This primer of pathology is intended to introduce investigators to the structure (morphology) of cancer with an emphasis on genetically engineered mouse (GEM) models (GEMMs). We emphasize the necessity of using the entire biological context for the interpretation of anatomic pathology. Because the primary investigator is responsible for almost all of the information and procedures leading up to microscopic examination, they should also be responsible for documentation of experiments so that the microscopic interpretation can be rendered in context of the biology. The steps involved in this process are outlined, discussed, and illustrated. Because GEMMs are unique experimental subjects, some of the more common pitfalls are discussed. Many of these errors can be avoided with attention to detail and continuous quality assurance.

Limited mouse necropsy.

Procurement of mouse tissues or organs is essential for complete verification of almost any phenotype. A proper necropsy can yield information that is difficult to obtain by limited biopsy or surgical intervention. The protocol described here is for a limited autopsy involving the thorax and abdomen only, and does not include all organs.

Mouse tissue fixation.

One of the primary goals of fixation is to stop postmortem changes that degrade the tissue and allow optimal preservation of morphologic and cytological detail as well as nucleic acid integrity. Following death, tissues soon undergo autolysis, and if organisms from the gastrointestinal, urinary, or respiratory tracts are present, their colonization can soon cause putrefaction. Time is of the essence because warmer temperatures accelerate both types of degradation. Placing the tissue into a fixative stops the postmortem changes. Fixatives have their effect on tissue by cross-linking, coagulation, or a combination of both. This article outlines the basic tissue fixation procedure and offers guidance on choosing an appropriate fixative, the timing and duration of fixation, sample storage, and quality issues.

Structured reporting in anatomic pathology for coclinical trials: the caELMIR model.

Electronic media, with their tremendous potential for storing, retrieving, and integrating data, are an essential part of modern collaborative multidisciplinary science. Structured reporting is a fundamental aspect of keeping accurate, searchable electronic records. This discussion on structured reporting in anatomic pathology for pre- and coclinical trials in animal models provides background information for scientists who are not familiar with structured reporting. Practical examples are provided using a working database system for preclinical research-caELMIR (Cancer Electronic Laboratory Management Information and Retrieval)-developed by the U.S. National Cancer Institute's (NCI's) Mouse Models of Human Cancers Consortium (MMHCC).

Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers.

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.

Phosphatase and tensin homologue deleted on chromosome 10 deficiency accelerates tumor induction in a mouse model of ErbB-2 mammary tumorigenesis.

Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2(KI)) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2(KI) strain. Tumor progression in PTEN-deficient/ErbB-2(KI) strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2(KI)-derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2(KI) parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2-induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer.

Protective immunity to SIV challenge elicited by vaccination of macaques with multigenic DNA vaccines producing virus-like particles.

We utilized SIV(mne) infection of Macaca fascicularis to assess the efficacy of DNA vaccination alone, and as a priming agent in combination with subunit protein boosts. All SIV(mne) structural and regulatory genes were expressed using the human cytomegalovirus Immediate Early-1 promoter in plasmids that directed the formation of virus-like particles in vitro. Macaques (n = 4) were immunized intradermally and intramuscularly four times over 36 weeks with 3 mg plasmid DNA. A second group (n = 4) received two DNA priming inoculations followed by two intramuscular boosts consisting of 250 microg recombinant Env gp160 and 250 microg recombinant Gag-Pol particles in MF-59 adjuvant. These regimens elicited modest cellular immunity prior to challenge. Humoral immune responses to Env gp160 were elicited and sustained by both vaccine protocols, and as expected antibody titers were higher in the protein subunit-boosted animals. Neutralizing antibodies prior to challenge were measurable in two of four subunit-boosted macaques. The two vaccine regimens elicited comparable helper T cell responses at the time of challenge. Vaccinees and mock-immunized controls (n = 4) were challenged intrarectally at week 38 with uncloned SIV(mne). Following challenge all macaques became infected, but both vaccine regimens resulted in reduced peak virus loads (p = 0.07) and significantly improved maintenance of peripheral CD4(+) T cell counts postchallenge (p = 0.007, DNA alone and p = 0.01, all vaccinees). There was no significant difference between the two vaccine groups in levels of plasma viremia or maintenance of CD4(+) T cell counts postchallenge.

Molecular analysis of metastasis in a polyomavirus middle T mouse model: the role of osteopontin.

INTRODUCTION: In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential. METHODS: Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods. RESULTS: With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells. CONCLUSIONS: We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model.

Disseminated microsporidiosis in a pancreas/kidney transplant recipient.

Human microsporidiosis has been described most commonly in patients with acquired immunodeficiency syndrome and only rarely in those with other forms of immunosuppression. Only 11 cases of microsporidiosis have been reported previously in solid transplant recipients. To our knowledge, this is the first report to describe a case of microsporidiosis in a pancreas/kidney transplant recipient in whom multi-organ system dissemination was observed. This infection was not detected until postmortem examination of stained tissue sections revealed microsporidian spores that were identified as Encephalitozoon species by transmission electron microscopy. It is suspected that leakage from the duodenal anastomosis to the bladder may have contributed to the dissemination of this infection.

Follicular lipidosis in three Rottweilers.

Abstract Follicular lipidosis is reported in three young Rottweilers that developed hypotrichosis of the mahogany-coloured points of the face and feet. One dog had concurrent lightening and dulling of the remaining hair. Hair matrix cells were swollen with intracellular lipid, identified by electron microscopy and oil-red-O staining. Concurrent abnormalities in one dog included poor somatic growth and elevation in serum concentration of blood urea nitrogen and creatinine. Necropsy of this dog revealed small thyroid glands with no visible colloid production, and chronic renal disease. The two remaining dogs were otherwise healthy. One of these two dogs had partial resolution of the hair loss with persistence of mild histological changes of the hair follicles 7 months after first presentation. The second was lost to follow-up but written records had no mention of skin lésions at presentation for cruciate repair 3 years after initial recognition of the haircoat changes. To the authors' knowledge, this is the first report of follicular lipidosis in any species. Résumé- Une lipidose folliculaire est observée sur trois jeunes Rottweilers qui développent une hypotrichose des zones acajou de la face et des pieds. Un chien présente également un éclaircissement et un aspect terne des poils restant. Les cellules matricielles des poils sont gonflées par des lipides en position intracellulaire identifies par la microscopie électronique et une coloration au Rouge Congo. D'autres symptômes sont observes chez un chien: retard de croissance et des elevations de l'urémie et de la créatininémie. L'autopsie de ce chien a montré des glandes thyroidiennes petites et une insuffisance rénale chronique. Les deux autres chiens sont en bonnes santé. Un des deux chiens a présente une resolution partielle de l'hypotrichose avec une persistance modérée des modifications histopathologiques des follicules, sept mois après lä premiere visite. Le second a été perdu de vue mais des contacts par écrit ont permis de savoir que le chien ne présentait plus de lésions, trois ans après la premiere visite. A la connaissance de l'auteur, il s'agit de la premiere description de lipidose folliculaire. [Gross, T. L., Pascal-Tenorio, A., Munn, R. J., Hargis, A. M., Kline, A. Follicular lipidosis in three Rottweilers. (Lipidose folliculaire chez trois Rottweilers.) Veterinary Dermatology 1997; 8: 33-40.] Zusammenfassung- Bei drei jungen Rottweilern, die Hypotrichie an den mahagonifarbenen Stellen im Gesicht und an den Pfoten entwickelten, wird von follikulärer Lipidose berichtet. Einer der Hunde hatte gleichzeitig eine Aufhellung und Abstumpfung des übrigen Haarkleides. Haarmatrixzellen waren mit intrazellulärem Lipid angeschwollen, das mittels Elektronenmikroskopie und 'oil-Red-O' Färbung identifiziert wurde. Bei einem Hund waren gleichzeitig schlechtes somatisches Wachstum und erhoehte Harnstoff-und Kreatininspiegel im Serum feststellbar. Nekropsie dieses Hundes ergab kleine Schilddrüsen ohne wahrnehmbare Kolloidproduktion und chronische Nierenerkrankung. Die zwei anderen Hunde wareii ansonsten gesund. Das Haarkleid einer der beiden Hunde wuchs teilweise wieder, leichte histologische Veränderungen waren 7 Monate nach der ersten Unetersuchung immer noch vorhanden. Der zweite Fall wurde nicht weiterverfolgt, jedoch erwähnten die Krankenberichte 3 Jahre nach dem Beginn der Haarkleidveränderungen keine diesbezüglichen Anomalien. Soviel die Autoren wissen, ist dieses der erste Bericht über follikuläre Lipidose bei Tieren. [Gross, T. L., Pascal-Tenorio, A., Munn, R. J., Hargis, A. M., Kline, A. Follicular lipidosis in three Rottweilers. (Folliculäre Lipidose bei 3 Rottweilern.) Veterinary Dermatology 1997; 8: 33-40.] Resumen Se describió una lipidosis folicular en tres Rottweilers jóvenes que desarrollaron hipotricosis en los puntos color caoba de la cara y patas. Uno de los perros tenia a su vez el resto del pelo más claro y mate. Les células de la matriz del pelo se encontraban tumefactas, con lipido intracelular identificado medíante microscopia electrónica y tinción roja-O. Un perro presentaba concomitantement crecimiento somático deficiente y elevaciones séricas de BUN y creatinina. El estudio de necropsia en este perro revaló glándulae tiroides de pequeño tamaño, sin producción de coloide visible, asi como enfermedad renal crónica. Los otros dos animales se encontraban en buen estado de salud. Uno de ellos experimentó una resolución parcial de la pérdida de pelo con persistencia de alteraciones histológicas leves de los foliculos 7 meses después de la primera presentación. No fue posible el seguimiento del segundo perro, pero no se mencionaban lesiones cutáneas en el informe escrito del animal al presentarse 3 años después para una intervención de ligamentos cruzados, después de la alteraciones iniciales. Según nuestros datos, esta es la primera descripción de lipidosis folicular en cualquier especie. Gross, T. L., Pascal-Tenorio, A., Munn, R. J., Hargis, A. M., Kline, A. Follicular lipidosis in three Rottweilers. (Lipidosis folicular en tres Rottweilers.) Veterinary Dermatology 1997; 8: 33-0.].