Paraffin-embedded Mouse Tissues Research Articles

Development and characterization of rat monoclonal antibodies to denatured mouse angiotensin‐converting enzyme

Four new rat monoclonal antibodies, generated to denatured mouse somatic angiotensin-converting enzyme (ACE, CD143), detect mouse ACE with high sensitivity in Western blotting. Epitope mapping for the monoclonal antibodies--B12, 4G6 and 5C4--was also performed. Two monoclonal antibodies--B12 and 5C4--are directed to various epitopes on the N-domain--i.e., they recognized only the somatic isoform of mouse ACE. The monoclonal antibody H7 recognized an epitope on the C-domain of mouse ACE. The monoclonal antibody 4G6 was directed to a sequence on the N-domain of mouse ACE, which is homologous to a region of the C-domain and, as a result, also recognizes mouse testicular ACE (tACE) by means of Western blotting. In paraffin-embedded mouse tissues, all monoclonal antibodies detected all known expression sites of somatic ACE (sACE), e.g., the epithelial cells of the kidney proximal tubules, intestine and epididymis, and heterogeneously in endothelial cells. The monoclonal antibodies 4G6 and H7 additionally stained mouse tACE in spermatozoa and in mature spermatids. The monoclonal antibody 4G6 also demonstrated cross-reactivity with sACE from a broad spectrum of animal species, including human, rat, rabbit and bovine. However, this monoclonal antibody did not recognize the testicular isoform of ACE of these species. This set of monoclonal antibodies is useful for identifying even subtle changes in mouse ACE conformation because of denaturation. These monoclonal antibodies are also sensitive tools for the detection of mouse ACE in biological fluids and tissues by using proteomics approaches. Their high reactivity in paraffin-embedded tissues opens up opportunities to study possible changes in the pattern of ACE expression in knockout mouse models and may prove useful for correlating ACE expression in these models with human diseases.

Antibodies that label paraffin-embedded mouse tissues: a collaborative endeavor.

Histology and immunohistochemistry are important tools in the study of human diseases and their respective animal models. The study of mouse models has been hampered by the absence of a large set of mouse-specific antibodies adapted to paraffin-embedded tissues. A total of 196 antibodies were tested on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). The antibodies were targeted to proteins of the cytoplasm (n = 100), plasma membrane (n = 48), nucleus (n = 36), extracellular compartment (n = 5), cytoplasm/cell membrane (n = 4), and viral proteins (n = 3). A total of 83 antibodies provided an adequate signal to noise ratio. Of these, adequate labeling required heat-mediated epitope retrieval or enzymatic digestion for 32 and 8 antibodies, respectively. Epitope recognition was best for tissues fixed with Fekete's acid-alcohol-formalin. However, some proteins could be detected only in IHC Zinc Fixative, confirming that there is no single fixative suitable for the preservation of all epitopes. Four of 13 antibodies that failed to label their cellular targets on tissue sections successfully labeled whole-mount tissues, indicating that tissue processing plays an important role in epitope degradation. Regularly updated information on immunohistochemistry of normal and neoplastic mouse tissues is accessible online at (http://tumor.informatics.jax.org); links to antibody suppliers' web sites are provided.

A review of morphological techniques for detection of peroxisomal (and mitochondrial) proteins and their corresponding mRNAs during ontogenesis in mice: application to the PEX5-knockout mouse with Zellweger syndrome.

In the era of application of molecular biological gene-targeting technology for the generation of knockout mouse models to study human genetic diseases, the availability of highly sensitive and reliable methods for the morphological characterization of the specific phenotypes of these mice is of great importance. In the first part of this report, the role of morphological techniques for studying the biology and pathology of peroxisomes is reviewed, and the techniques established in our laboratories for the localization of peroxisomal proteins and corresponding mRNAs in fetal and newborn mice are presented and discussed in the context of the international literature. In the second part, the literature on the ontogenetic development of the peroxisomal compartment in mice, with special emphasis on liver and intestine is reviewed and compared with our own data reported recently. In addition, some recent data on the pathological alterations in the liver of the PEX5(-/-) mouse with a peroxisomal biogenesis defect are briefly discussed. Finally, the methods developed during these studies for the localization of mitochondrial proteins (respiratory chain complexes and MnSOD) are presented and their advantages and pitfalls discussed. With the help of these techniques, it is now possible to identify and distinguish unequivocally peroxisomes from mitochondria, two classes of cell organelles giving by light microscopy a punctate staining pattern in microscopical immunohistochemical preparations of paraffin-embedded mouse tissues.

Detection of Candida albicans mRNA from formalin-fixed, paraffin-embedded mouse tissues by nested reverse transcription-PCR.

Histopathology archives represent a vast source of infectious disease specimens that can be used to elucidate important disease processes. In this report, we describe a method to detect Candida albicans gene expression from infected, formalin-fixed, paraffin-embedded mouse tissue. By use of glass beads to break open the fungal cells and proteinase K treatment, RNA was extracted routinely from tissue sections that had been fixed for up to 72 h. Upon reverse transcription of the RNA and nested PCR, the procedure detected C. albicans "housekeeping" and putative virulence genes.

Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues

We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10, IFN γ and TNF α using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate–lysine–paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54°C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNF α, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFN γ. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.