Journal of Histochemistry & Cytochemistry

Ribrioux, S; Kleymann, G; Haase, W; Heitmann, K; Ostermeier, C; Michel, H

doi: 10.1177/44.3.8648079pmid: 8648079

Recombinant antibody fragments are emerging as a versatile tool in both basicresearch and medical therapy. We describe the procedures for direct labeling ofengineered antibody fragments (Fv) with fluorescein or nanogold and their use influorescence and immunoelectron microscopy, respectively. The Fv fragments wereproduced in Escherichia coli, purified by one-step Strep tag affinity chromatography,chemically labeled with the marker, and employed in microscopy to localize epitopeson the membrane protein bacteriorhodopsin in purple membranes of Halobacteriumhalobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases,methods involving directly labeled antibody fragments show results identical to thosein which antibodies or Fv fragments are detected by a secondarily labeled conjugate.The multifunctional design of the recombinant Fv fragments, however, offers moreall-around applications in immunocytochemistry. The directly labeled Fv fragments,half the size of an Fab fragment, are at the molecular level the smallest antibodyfragments yet described for visualization of biomolecules in microscopy.

Jeraldo, T L; Coutu, J A; Verdier, P A; McMillan, P N; Adelson, J W

doi: 10.1177/44.3.8648080pmid: 8648080

Homogeneity in structure and function are broadly assumed to be characteristics of the acinar pancreatic digestive enzyme-secreting tissue. In recent years, physiological studies have shown that the pancreas stores the digestive enzymes in heterogeneously composed pools and releases them from these pools in a cyclic and secretagoguec fashion. The cellular basis for pancreatic heterogeneity is unknown; classical light and electron microscopic preparations appear homogeneous. We applied a panel of biotinylated lectins to pancreatic tissue sections; acinar cell glycoconjugates were localized in situ with peroxidase and fluorescent techniques and lectin-gold complexes. The lectin-binding properties of both fasting rabbit and rat pancreas revealed extensive and specific heterogeneity of the acinar cell population. Light and electron microscopy demonstrated highly heterogeneous labeling of the zymogen granule contents of specific acinar cells with the lectins Ulex europaeus agglutinin (UEA) and Erythrina cristagalli (ECA), which also showed preferential labeling of peri-insular acini. Other lectins also demonstrated heterogeneous binding to specific cellular regions. The striking acinar cell heterogeneity confirms earlier predictions, and may eventually prove to be the cellular basis for the secretion of different enzyme mixtures from heterogeneous sources within the pancreas.

Gown, A M; Jiang, J J; Matles, H; Skelly, M; Goodpaster, T; Cass, L; Reshatof, M; Spaulding, D; Coltrera, M D

doi: 10.1177/44.3.8648081pmid: 8648081

Several different methods of measuring proliferation indices have been developed, including measurements of cellular DNA content (flow cytometry), S-phase incorporation of thymidine analogues into DNA (e.g., tritiated thymidine and 5'-bromodeoxyuridine), and immunostaining of cell cycle-restricted proteins (e.g., Ki-67 antigen and PCNA). Theoretical and practical problems with each method have made it difficult to compare absolute proliferation rates among cells of different lineages and degrees of malignancy. More recently, in situ hybridization (ISH) for histone 3 (H3) mRNA has been introduced. We used a double labeling method for comparing H3 mRNA expression and S-phase incorporation of 5'-bromodeoxyuridine (BrdU) to determine if H3 mRNA expression was tightly associated with S-phase in a variety of malignant and nontransformed cell types. In addition, labeling results were compared in methacarn- and formalin-fixed tissues to extend the potential usefulness of H3 ISH, using a postfixation technique for the alcohol-fixed specimens. As expected for a cumulative marker, variation was noted in the percentage of the BrdU-positive cells double labeled with H3 ISH (53-89%), depending on cell type and length of BrdU incubation. In contrast, the percentage of the H3 ISH-positive cell population double labeled for BrdU was independent of the cell type of BrdU incubation time (mean 78%). Similarly, a consistent percentage of H3 ISH-positive cell populations was double labeled for BrdU in normal tissues (mean 97%). These findings support a well-conserved timing mechanism for H3 mRNA expression and DNA replication. We conclude that H3 ISH is an extremely accurate technique for assessment of S-phase cell proliferation indices.

Huitfeldt, H S; Skarpen, E; Lindeman, B; Becher, R; Thrane, E V; Schwarze, P E

doi: 10.1177/44.3.8648082pmid: 8648082

Transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) are strong hepatocyte mitogens and important regulators of liver regeneration. The TGF-alpha receptor EGFr appears primarily to mediate a proliferative signal, whereas mitogenic, motogenic, and morphogenic effects have been attributed to activation of the HGF receptor Met. We have studied the localization of Met and EGFr in normal and carcinogen-treated rat livers. Oval cells and preneoplastic lesions were induced by diethylnitrosamine initiation, followed by promotion with 2-acetylaminofluorene combined with a partial hepatectomy. Different liver cell populations and their receptor expression were characterized by two-color immunofluorescence and confocal laser scanning microscopy. Hepatocytes were detected by keratin K8 staining, and oval cells and bile ducts were recognized by keratin K19 expression. Enzyme-altered preneoplastic lesions ere identified by expression of placental glutathione S-transferase (GST-pi). Staining for these cellular markers was combined with immunodetection of EGFr and Met. Normal liver exhibited strong staining for EGFr in hepatocytes, whereas blood vessels, bile ducts, and some sinusoidal cells were Met-positive. In carcinogen-treated livers, oval cells showed Met but not EGFr immunostaining. GST-pi-positive foci displayed EGFr immunostaining at a similar intensity as surrounding hepatocytes, whereas Met was not detected. Our data indicate that putative liver cells (oval cells) have a growth receptor phenotype similar to that of bile ducts, whereas preneoplastic live lesions appear hepatocyte-like. These results indicate that the preferential proliferation of preneoplastic liver lesions compared to surrounding hepatocytes is not associated with an altered EGFr or Met phenotype.

Janckila, A J; Lear, S C; Martin, A W; Yam, L T

doi: 10.1177/44.3.8648083pmid: 8648083

We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.

Parkkila, A K; Parkkila, S; Rajaniemi, H

doi: 10.1177/44.3.8648084pmid: 8648084

We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.

Fukui, M; Fujimoto, T; Watanabe, K; Endo, K; Kuno, K

doi: 10.1177/44.3.8648085pmid: 8648085

It was recently found that certain cells in the alveolar septum of the bovine lung are enriched in prostaglandin F (PGF) synthase. In this study we used immunohistochemical techniques at both light and electron microscopic levels to further characterize the PGF synthase-positive cells. By double immunofluorescence staining of bovine lung cryostat sections, the alveolar septal cells labeled by anti-PGF synthase antibody were also intensely labeled for cytoplasmic actin but not for alpha-smooth muscle actin. This labeling pattern suggests that the PGF synthase-positive cells in the septum are "contractile interstitial cells," which resemble conventional fibroblasts but characteristically contain prominent bundles of actin filaments. Immunogold electron microscopy of ultra-thin frozen sections of bovine lung showed that alveolar interstitial cells extending long cytoplasmic processes and closely associated with alveolar capillaries were intensely labeled for PGF synthase. Capillary endothelial cells, alveolar epithelial cells, and some fibroblastic cells were devoid of labeling. On the basis of these findings, we conclude that PGF synthase is specifically expressed in contractile interstitial cells within the alveolar septum. The protein may be a useful marker for contractile interstitial cells, whose physiological function and role in various pathological conditions have not been characterized in detail.

Holland, M S; Mackenzie, C D; Bull, R W; Silva, R F

doi: 10.1177/44.3.8648086pmid: 8648086

Our objective was to identify an optimal single set of conditions for use in both indirect immunofluorescence assays (IFA) and in situ hybridization (ISH) to detect viral proteins and nucleic acids in avian lymphoid and neural tissues. Various fixatives were evaluated for use with IFA to detect turkey Herpesvirus (HVT) glycoprotein B (gB) and ISH to identify HVT mRNA in chicken tissues. A precipitating fixative (acetone) was compared to crosslinking fixatives [buffered glutaraldehyde-picric acid (BGPA), 10% formalin, and 4% paraformaldehyde] for both IFA and ISH using spleen, thymus, bursa, sciatic plexus, and brachial plexus of 28-day-old chickens. Four percent paraformaldehyde was found to be the optimal fixative for preservation of all chicken tissues examined with both IFA and ISH. Glass slide preparation, incubation temperatures, and tissue processing were each individually evaluated for ISH and IFA. Silylated slides provided the best retention of tissue sections for both procedures. For IFA, 37 degrees C was the ideal incubation temperature tested, whereas the optimal incubation temperature tested for ISH was 47 degrees C. Of the blocking agents compared, Evans blue dye prevented background fluorescence to a greater extent than either calf serum or bovine serum albumin. These findings provide a technical basis for investigations into various aspects of the molecular pathology of avian diseases.

Vult von Steyern, F; Josefsson, J O; Tågerud, S

doi: 10.1177/44.3.8648087pmid: 8648087

We describe a very efficient method for fluorescent labeling of acidic structures in denervated skeletal muscle with rhodamine B. Rhodamine B at 50 ng/ml gave selective and distinct segmental labeling of denervated muscle fibers after 5-min incubation at room temperature. Labeling was also achieved at 4 degrees C. The labeling was disrupted by the ionophores monensin and nigericin, suggesting a labeling confined to acidic structures. Rhodamine B co-localized with the lysosomotropic dye Lyso Tracker Green and a marker for endocytosis (fluorescein isothiocyanate-labeled dextran). Rhodamine B, which is highly lipophilic, showed pH-dependent fluorescence emission in saturated aqueous N-octanol. Tetramethylrhodamine showed similar characteristics for labeling of denervated muscle fibers and pH-dependent fluorescence in N-octanol. The carboxyl group present in these two compounds appears important, because structurally related compounds that either lack this group or have it esterified failed to label denervated muscle fibers and showed no pH-dependent fluorescence in N-octanol. The results suggest that rhodamine B labels acidic organelles belonging to the endosomal/lyosomal system of denervated skeletal muscle fibers. Nevertheless, it failed to label such organelles in a number of mammalian cell types other than denervated skeletal muscle fibers.

Seiffert, D

doi: 10.1177/44.3.8648088pmid: 8648088

Adhesive glycoproteins in the bone matrix are of critical importance for cell anchorage, proliferation, migration, differentiation, and regulation of bone metabolism. The localization of the adhesive glycoprotein vitronectin (Vn) in murine bone tissue was evaluated by immunohistochemical staining. Vitronectin was present throughout the mineralized bone matrix of cancellous and cortical bone, whereas cartilage was devoid of Vn staining. To exclude the possibility that the positive Vn staining resulted from plasma Vn in blood vessels within the bone sections, adjacent tissue sections were stained with antibodies to fibrinogen, and abundant plasma protein. Fibrinogen immunoreactivity was confined to blood vessels in the bone marrow and Haversian system, whereas the mineralized bone matrix was devoid of staining. The presence of Vn in murine bones was confirmed by sequential extraction, followed by fractionation of the resulting polypeptides by gel electrophoresis and immunoblotting analysis. Hydroxyapatite affinity chromatography raises the possibility that mineral interactions, at least in part, mediate the incorporation of Vn into the bone matrix. These results indicate that Vn is a specific component of bone tissue and raise the possibility that Vn is involved in regulation of bone metabolism.