bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.
Authors: Beckstead J H
Published: April 2011

Immunohistochemistry is a powerful tool for tissue diagnosis and research. Although the frozen section has remained the gold standard for this important approach to evaluation of antigens in tissues, there is widespread acknowledgment of many limitations. Routine paraffin-embedded sections ware widely used for morphological examination of tissues but are not optimal for antigen preservation. In this study, paraffin-embedded tissues fixed with a simple buffer containing zinc as the primary fixative were compared with tissues fixed with routine formalin, zinc-formalin, paraformaldehyde, ethanol, a variety of commercial (non-formalin-containing) fixatives that have been recommended for reduced toxicity and improved antigen survival, and frozen sections. Human lymphoid tissues and a group of antibodies to antigens (CD1, CD4, CD7, CD8, CD19) usually preserved only in frozen tissue were used as a model system. Fixation in a simple solution of zinc acetate and zinc chloride in a Tris-Ca acetate buffer resulted in antigen preservation comparable to that in frozen sections with antibodies to these cell surface markers. Morphological preservation was comparable to formalin-fixed sections. The work presents a new method that represents the closest approach yet to a technique that combines optimal antigenic survival with the convenience and morphological preservation of traditional formalin-fixed tissue embedded in paraffin.
Immunocytochemical localization of Lewis blood group antigens in human salivary glands.
Authors: Cossu M , Lantini M S , Puxeddu R
Published: April 2011

We demonstrated the immunohistochemical distribution of Le-a and Le-b blood group antigens in human major and minor salivary glands at the ultrastructural level by applying a post-embedding immunogold staining method. In secretors' glands, a faint Le-a reactivity was found only in mucous droplets, whereas Le-b antigen was intensely stained in secretory granules of most mucous cells, in those of intercalated duct cells, in the pale granular matrix of some serous cells, and, when osmication was omitted, in cytoplasmatic vesicles and cell surfaces of striated ducts. In the submandibular gland of a non-secretor, Le-a antigen was considerably stained in mucous droplets, whereas Le-b reactivity was restricted to the striated duct cells. These results indicate that the secretor status affects the secretion of Lewis antigens by mucous, serous, and intercalated duct cells but not the presence of Le-b as a surface antigen in striated duct cells.
Temporal-spatial distribution of SP-B and SP-C proteins and mRNAs in developing respiratory epithelium of human lung.
Authors: Khoor A , Stahlman M T , Gray M E , Whitsett J A
Published: April 2011

We determined the temporal and spatial distribution of surfactant protein B (pro-SP-B) and C (pro-SP-C) mRNAs and proteins by immunohistochemistry and in situ hybridization in fetal, neonatal, and adult human lung. Pro-SP-B and SP-B mRNA were detected in bronchi and bronchioles by 15 weeks' gestation. After 25 weeks, pro-SP-B, active SP-B peptide, and SP-B mRNA were co-localized in bronchiolo-alveolar portal cells and in Type II epithelial cells. In adult lung, pro-SP-B and SP-B mRNA were detected primarily in non-ciliated bronchiolar epithelial cells and in Type II cells in the alveolus. Pro-SP-C and SP-C mRNA were detected in cells lining terminal airways from 15 weeks' gestation and thereafter. After 25 weeks, SP-C mRNA and precursor protein were detected in epithelial cells of the bronchiolo-alveolar portals and in Type II cells, where expression increased with advancing gestational age. Distinct cellular patterns of staining for pro-SP-B compared with SP-B active peptide support the concept that its proteolytic processing or cellular routing may be influenced by cell type and/or cell differentiation. SP-B and SP-C are expressed primarily in distal conducting and terminal airway epithelium of human fetal lung well in advance of surfactant lipid synthesis or physiologic requirements to produce pulmonary surfactant at the time of birth.
The alpha 6 and beta 4 integrin subunits are expressed by smooth muscle cells of human small vessels: a new localization in mesenchymal cells.
Authors: Cremona O , Savoia P , Marchisio P C , Gabbiani G , Chaponnier C
Published: April 2011

The alpha 6 beta 4 integrin complex is generally thought to be expressed by epithelial cells, where it is localized in specific adhesion structures, the hemidesmosomes. Recent observations have suggested a new localization of the beta 4 integrin chain in small vessels, possibly in endothelial cells, i.e., in cells of mesenchymal origin. In the present study we show that (a) the alpha 6 and beta 4 integrin chains are not expressed by endothelial cells, since they are not localized in von Willebrand factor-producing cells; (b) instead, smooth muscle cells of small vessels are intensely positive to antibodies to both alpha 6 and beta 4 intergrin chains; and (c) in some restricted regions of these smooth muscle cells there is a clear colocalization between alpha-smooth muscle actin and alpha 6 and beta 4 integrin chains, suggesting that a new type of cytoskeletal linkage for the alpha 6 beta 4 integrin complex may occur in mesenchyme-derived cells. Our observations are supported by confocal laser microscopy (CLSM) images of specimens labeled by double immunofluorescence. This technical choice was made to take advantage of the higher resolution offered by CLSM in comparison with conventional immunofluorescence. A careful selection of barrier filters was necessary to separate accurately emission and excitation spectra of the fluorochromes used in this study, resulting in an efficient colocalization analysis.
Immunohistochemical localization of carbonic anhydrase IV in capillaries of rat and human skeletal muscle.
Authors: Sender S , Gros G , Waheed A , Hageman G S , Sly W S
Published: April 2011

We used polyclonal antisera raised in rabbits against membrane-bound rat lung and human lung carbonic anhydrase (CA) IV in immunofluorescence studies to stain cryosections of rat soleus and extensor digitorum longus (EDL) and several human skeletal muscles. There was strong specific staining of capillaries in all muscles investigated. Several techniques were applied to verify this result. (a) Serial sections were either incubated with anti-CA IV/FITC or processed for endothelial ATPase reaction. There was precise co-localization of antibody marked structures and ATPase stained capillaries. (b) Human muscle sections were double stained with anti-CA IV/TRITC and anti-von Willebrand factor (vWF)/FITC. vWF, a capillary marker, and CA IV were localized at identical sites. (c) The CAIV was released from capillaries by treatment with phosphatidylinositol specific phospholipase C, suggesting that the enzyme is anchored to the endothelial cell membrane via a phosphatidylinositolglycan anchor. (d) A rat hindlimb was perfused with diluted antiserum. Cryosections of perfused soleus and EDL processed for anti-rabbit IgG/FITC staining showed clear fluorescence associated with capillaries, indicating that the antigen was accessible from the capillary lumen. (e) Immune complexes formed during antiserum perfusion as described in d were precipitated from muscle homogenates. SDS-PAGE followed by immunoblotting showed that the predominant portion of total muscle CA IV was bound in these complexes and therefore must be located intravascularly.
Potential problems in using [35S]-dATP-tailed oligonucleotides for detecting mRNAs in certain cells of the immune system.
Authors: Mezey E , Hoffman B J , Harta G , Palkovits M , Northup J
Published: April 2011

In this study we examined the cause of unusually intense signals obtained in immune cells by in situ hybridization histochemistry using 35S-labeled oligonucleotides. We verified that the phenomenon is an amplification of a specific signal due to a series of chemical interactions after the probe binds to a specific mRNA in the tissue. The presence of oxidative enzymes in the tissue seems to be necessary for this reaction to occur. Therefore, most cells of the immune system (e.g., macrophages, neutrophil and eosinophil leukocytes), being rich in oxidative enzymes, will show some signal amplification. The intensification of the signal can be avoided if MgCl2 is substituted for CoCl2 in the synthesis of [35S]-thiophosphate-labeled probes, if 2,3-dimercaptopropanol [British anti-Lewisite (BAL)] is added to the hybridization buffer, or if [33P]-phosphate is used instead of [35S]-thiophosphate in the labeling of the probes.
Calibration of 14C-plastic standards for quantitative autoradiography with 33P.
Authors: Eakin T J , Baskin D G , Breininger J F , Stahl W L
Published: April 2011

Probes labeled with 33P have potential for widespread use in in situ hybridization because they are better able to detect relatively scarce mRNAs compared with probes labeled with 35S, but the relatively short half-life of 33P is a disadvantage when it is used as a radioactivity standard for quantitative autoradiography. To determine if plastic sections containing 14C can be used as standards for quantitative autoradiography with 33P, we co-exposed 33P-labeled liver paste sections and 14C-plastic standards to Hyperfilm beta max. The autoradiographic response of Hyperfilm beta max to these isotopes was almost identical. Second-order polynomial equations obtained from analysis of film relative optical density and radioactivity permitted derivation of tissue-equivalent radioactivity from the film optical densities produced by the 14C standards for 1-14-day exposures. These results validate the use of plastic 14C standards for quantifying 33P used in contact film autoradiography.
The SA/rABC technique: a new ABC procedure for detection of antigens at increased sensitivity.
Authors: Grumbach I M , Veh R W
Published: April 2011

Since its introduction, the avidin-biotin-peroxidase (ABC) complex has become an invaluable detection system for a wide variety of bioanalytical applications. In these techniques, avidin and biotin-peroxidase are mixed at a pre-determined ratio so that the soluble ABC complex retains biotin binding sites. Consequently, the complex contains an excess of avidin over biotinylated peroxidase residues. On theoretical considerations, however, an ABC complex designed for maximal signal intensity must consist of an excess of peroxidase over avidin molecules. Therefore, ABC complexes with reversed molar ratios of biotinylated peroxidase to avidin (rABC complexes) were prepared and an intermediate streptavidin step was introduced to bind the rABC complexes to biotinylated IgG molecules. The signal generating power of this new streptavidin-rABC sequence proved superior to that of the conventional ABC technique in ELISA assays and in immunostaining of tissue sections.
Simultaneous immunoenzymatic staining of catecholamines, catecholamine-biosynthesizing enzymes, and bromodeoxyuridine in adrenal medullary cells of the rat.
Authors: Ubink R , Lange W , Verhofstad A
Published: April 2011

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.
In situ hybridization of parathyroid hormone-related protein in normal skin, skin tumors, and gynecological cancers using digoxigenin-labeled probes and antibody enhancement.
Authors: Danks J A , McHale J C , Clark S P , Chou S T , Scurry J P , Ingleton P M , Martin T J
Published: April 2011

We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-embedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from barely to highly discernible and interpretable, with virtually no nonspecific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHrP mRNA expression was confined to the basal and spinous keratinocyte layers of skin. There was strong hybridization in the spinous keratinocyte layer and a low level of hybridization in the basal layer. An extensive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcinomas and basal cell carcinomas of the skin, from pathology archives, were examined for the presence of PTHrP mRNA. The results reflected previous immunohistochemical studies, with every squamous cell carcinoma hybridizing strongly with the PTHrP probes. The basal cell carcinomas showed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynecological tract also reflected the immunohistochemical findings, with expression found in the squamous cell carcinomas but not in the adenocarcinomas. In situ hybridization with digoxigenin-labeled oligonucleotide probes and antibody enhancement has provided a sensitive, highly specific procedure for detection of PTHrP mRNA in tumors and normal tissue.
Immunohistochemical localization of rK9, an enzyme of the kallikrein gene family, in the rat ventral prostate.
Authors: Berg T , Schøyen H
Published: April 2011

The rat kallikrein family consists of multiple closely related proteins. Most of these proteins have been purified from rat submandibular gland (SMG), among them rK9, an enzyme that has a direct vasoconstrictory effect on isolated aortic rings. Recently, two members of the kallikrein family, rK8 and rK9, were also purified from rat prostate, whereas other family members were not detected in this organ. In the present study, the immunohistochemical localization of rK9 was determined in rat prostate by the indirect immunofluorescence technique with polyclonal antisera against rat SMG rK9 in the first layer. The crossreactivity pattern of the antiserum against rat prostate tissue extract was established by flat-bed isoelectrofocusing and immunoblotting. The antiserum reacted with prostate rK9 but not with prostate rK8. Specificity of the immunofluorescent staining reaction was verified by staining with the primary antiserum after addition of purified SMG or prostate rK9, or other members of the kallikrein family including prostate rK8. rK9-specific immunofluorescence was detected in the prostate ductal structures. In the distal (acinar) segments, rK9 was found in the cytoplasm as granular staining in the middle third of the cell, in the luminal cell wall, and in the secretion within the lumen. In the intermediate segments, strong rK9-specific staining was observed adhering to the luminal wall as well as abundantly within the ductal lumen.
Immunocytochemical observation of the 90 KD heat shock protein (HSP90): high expression in primordial and pre-meiotic germ cells of male and female rat gonads.
Authors: Ohsako S , Bunick D , Hayashi Y
Published: April 2011

We used immunocytochemistry to detect the 90 KD major heat shock protein (HSP90), a potential regulator of gene expression, during male and female rat gonad development. In the Day 13.0 post-coital (dpc) fetal gonad, strong immunoreactivity to anti-HSP90 antibody was shown in the cytoplasm of primordial germ cells (PGCs). Other somatic cells in the gonad showed only faint reactivity. During testicular development, strong immunostaining was observed in the cytoplasm of embryonic germ cells and in spermatogonia and spermatocytes of the pre-pubertal testis. In adult testis reactivity of spermatogonia and pachytene spermatocytes was strong but reactivity of post-meiotic spermatogenic cells, i.e., secondary spermatocytes and spermatids, was extremely reduced. During ovarian development, immunostaining was also observed in the oogonia and the oocytes of pre-pubertal ovary. However, the staining of oocytes was reduced with the development of primordial follicles during the first week after birth. This study revealed that HSP90 is highly expressed in PGCs and continues to be expressed in both male and female pre-meiotic germ cells. The HSP90 accumulation may be essential for both male and female mammalian pre-meiotic germ cells.
A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens.
Authors: Lan H Y , Mu W , Nikolic-Paterson D J , Atkins R C
Published: April 2011

We report a simple and reliable method for detection of two or more antigens within tissue sections by indirect immunoenzyme staining using mouse monoclonal antibodies (MAbs). This technique involves treating sections with two 5-min microwave (MW) oven heatings between sequential rounds of three-layer immunoenzyme staining (mouse MAb, goat anti-mouse IgG, and mouse PAP or mouse APAAP) and color development. Discrete staining of cell surface, cytoplasmic, and nuclear antigens was evident within individual cells. This technique has a number of advantages over those currently available. First, MW treatment denatures bound antibody molecules, thereby completely blocking crossreactivity between sequential rounds of staining. This allows the use of primary (and other) antibodies raised in the same species and the use of a sensitive three-layer staining method. Second, antigen retrieval after MW treatment markedly increases the sensitivity of cytoplasmic and nuclear antigen detection. Third, inactivation of peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes prevents inappropriate color development. Finally, this method can be used in both paraformaldehyde-fixed cryostat sections and formalin-fixed paraffin tissue sections. In conclusion, this is a simple, reliable, and sensitive technique that will be useful in many areas of diagnosis and research.
Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.
Authors: Macville M V , Wiesmeijer K C , Dirks R W , Fransen J A , Raap A K
Published: April 2011

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.
Paraformaldehyde fixation of neutrophils for immunolabeling of granule antigens in cryoultrasections.
Authors: Elliott E , Dennison C , Fortgens P H , Travis J
Published: April 2011

Paraformaldehyde (PFA) fixation was optimized to facilitate the immobilization and labeling of multiple granule antigens, using short fixation regimens and cryoultramicrotomy of unembedded neutrophils (PMNs). In the optimal protocol, extraction of azurophil granule antigens (especially of the abundant elastase) was obviated by manipulating the polymeric state of PFA, and hence its rate of cross-linking, by altering its concentration and pH in a multistep process. Primary fixation conditions used (4% PFA, pH 8.0, 5 min) favor fixative penetration and rapid cross-linking. Stable cross-linking of the antigen was achieved in a secondary fixation step using conditions that favor larger, more cross-linking polymeric forms of PFA (8% PFA, pH 7.2, 15 min). Immobilization of granule antigens was enhanced by flotation of cut sections on fixative (8% PFA, pH 8.0) before labeling and by using post-labeling fixation with 1% glutaraldehyde. The optimized protocol facilitated immobilization and immunolabeling of elastase, myeloperoxidase, lactoferrin, and cathepsin D in highly hydrated, unembedded PMNs.
Localization of NADPH diaphorase in the mouse uterus during the first half of pregnancy and during an artificially induced decidual cell reaction.
Authors: Moorhead C S , Lawhun M , Nieder G L
Published: April 2011

Uteri from non-pregnant and pregnant (Days 1-10) mice were examined for the presence of NADPH diaphorase (NADPH-d) activity by histochemical techniques. Macrophages positive for NADPH-d were observed in all uterine sections but appeared to migrate out of the implantation site and cluster in the mesometrium and interimplantation zones beginning on Day 4. NADPH-d activity was seen in the luminal and glandular epithelium and in several isolated fibers coursing through the myometrium. Many branches of the uterine artery also expressed activity, with the most intense staining in the vessels of the mesometrium. However, the most remarkable staining began on Day 6 within the primary decidual zone. When the stromal cells underwent decidualization, they began to show NADPH-d activity, with the pattern of activity matching the expanding area of decidualization. By Day 9 most of the decidual cell reaction had occurred and the mesometrial decidual staining began to decrease. However, the blood vessels and the cells surrounding the developing blood spaces continued to express activity, and heavy staining was evident within the antimesometrial decidua. No NADPH-d activity was seen in any of the trophoblast cells at any time, or in embryonic tissue, except on Day 8. NADPH-d has been used to identify nitric oxide (NO) synthase. Therefore, it may represent an NO-mediated paracrine control over decidual blood flow, myometrial quiescence, or immune response during pregnancy.
A simple technique for staining of cell membranes with imidazole and osmium tetroxide.
Authors: Thiéry G , Bernier J , Bergeron M
Published: April 2011

We describe a simple new technique based on the affinity of imidazole and osmium tetroxide for unsaturated lipids. Organs (e.g., kidney, liver, intestine) were perfused in vivo with a glutaraldehyde solution. Tissue fragments were then immersed in a solution containing imidazole and OsO4 and are further stained with a double lead and copper citrate solution. Ultra-thin (0.06 microns) or thick (0.1-0.3 microns) sections were observed with transmission electron microscopy (80-100 kV). The method presented permits excellent visualization of cell membranes (e.g., endoplasmic reticulum, endocytotic apparatus) because it favors good resin penetration and the alkaline pH preserves cell volume. A better stereomicroscopic analysis of the relationship between cell organelles can be carried out with thick sections. The imidazole/osmium can be used routinely because the technical steps are easy and simple to follow. Furthermore, it can complement other cytochemical methods.
Ultrastructural localization of collagen types II, IX, and XI in the growth plate of human rib and fetal bovine epiphyseal cartilage: type XI collagen is restricted to thin fibrils.
Authors: Keene D R , Oxford J T , Morris N P
Published: April 2011

The collagen fibrils of hyaline cartilage vary in diameter depending on developmental stage and location within the tissue. In general, growth plates and fetal epiphyseal cartilages contain fibrils with diameters of less than approximately 25 nm, whereas the permanent cartilage of adult tissues contains fibrils of approximately 30-200 nm. The interstitial collagen fibrils of fetal cartilage are complex, having at least three collagen types as integral components. Type XI, a member of the fibrillar collagen class, has been proposed to limit fibril diameter. To test this proposition we sought to determine if Type XI collagen was preferentially associated with fibrils of smaller diameter. We focused our study on human juvenile rib growth plate, which has thin fibrils in the hypertrophic zone, thick fibrils in the resting zone or permanent cartilage, and a mixture of thin and thick fibrils in the proliferative zone. Tissues were examined by immunoelectron microscopy with antipeptide antibodies to the carboxyl telopeptide and to the amino terminal non-triple-helical domains of alpha 1 (XI). These studies showed that (a) both epitopes of Type XI collagen were readily accessible to antibodies at the fibrillar surface, (b) Type XI collagen was associated predominantly with fibrils < 25 nm in diameter, (c) Type XI collagen was not found in thick fibrils even after disruption with chaotropic agents, and (d) collagen Types II and IX were associated with fibrils of all sizes. These studies were extended to human newborn epiphyseal cartilage and to fetal calf cartilage, with the same result.
Immunogold localization of beta 1-integrin in bone: effect of glucocorticoids and insulin-like growth factor I on integrins and osteocyte formation.
Authors: Gohel A R , Hand A R , Gronowicz G A
Published: April 2011

Insulin-like growth factor I (IGF-I) and high-dose glucocorticoids exert opposite effects on bone formation. Because integrins are involved in cell and matrix organization, the effect of glucocorticoids and IGF-I on integrins was investigated in bone. An immunogold transmission electron microscopic (TEM) method was developed and applied to an organ culture system of 20-day fetal rat parietal bones, which mineralize in vitro. In parietal bone culture, 100 mM corticosterone treatment for 72 hr decreased calcification by 29%, disrupted osteoblast organization, and decreased the number of osteocytes. The quantity of osteoblast processes and the number of osteocytes per unit bone area were decreased by 48% and 56%, respectively. This effect was dose-dependent. The beta 1-integrin subunit was localized equally to apical and basal osteoblast surfaces by immunogold TEM. Compared to untreated control cultures, corticosterone (100 nM) decreased beta 1 by one third. In contrast, treatment with IGF-I for 72 hr increased calcification by 38%, cell processes by 71%, and osteocytes per unit area of bone by 107%. The number of gold particles localizing beta 1 on the osteoblast plasma membrane doubled, almost entirely on the apical surface of the osteoblast. Glucocorticoids and IGF-I had no significant effect on beta 1 levels in osteocytes. In conclusion, glucocorticoids and IGF-I modulate integrin levels on osteoblasts, and influence osteocyte formation and bone calcification. However, neither glucocorticoids nor IGF-I alter beta 1-integrin levels on osteocytes.
Patterns of immunoreactivity to an anti-fibronectin polyclonal antibody in formalin-fixed, paraffin-embedded oral tissues are dependent on methods of antigen retrieval.
Authors: Mighell A J , Robinson P A , Hume W J
Published: April 2011

The influence of antigen retrieval (AR) technique on immunoreactivity in formalin-fixed, paraffin-embedded tissues is recognized. In focal reactive overgrowths of oral mucosa, we noted that patterns of immunoreactivity to a fibronectin polyclonal antibody were dependent on methods of AR. To establish these patterns we investigated eight pyogenic granulomas and eight fibroepithelial polyps. In the absence of AR no immunoreactivity was observed. After alpha-chymotrypsin AR a band of intense immunoreactivity was associated with vascular endothelial cells, with either minimal or no staining of connective tissue. After microwave AR immunoreactivity was observed in connective tissue, especially in the subepithelial region, but there was no specific vascular staining. After autoclave AR immunoreactivity was observed in connective tissue and also in epithelial nuclei. To determine if these results represented either changes induced by tissue fixation and processing or exposure of epitopes normally masked in vivo, we investigated frozen sections from two fibroepithelial polyps and one pyogenic granuloma. In frozen sections immunoreactivity was observed around vascular endothelial cells and within connective tissue, especially in the subepithelial region, but not in epithelia. This study provides indirect evidence that alpha-chymotrypsin and heat-mediated AR protocols expose different masked epitopes, and reinforces the need to undertake appropriate pilot studies for immunohistochemical investigation of archival tissue.
Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain.
Authors: Lucassen P J , Chung W C , Vermeulen J P , Van Lookeren Campagne M , Van Dierendonck J H , Swaab D F
Published: April 2011

In situ end-labeling (ISEL) identifies DNA fragmentation in apoptotic or necrotic nuclei in tissue sections. However, application of ISEL on human brain requires conservation of DNA integrity during the postmortem delay (PMD) and good accessibility of fragmented DNA after (prolonged) tissue fixation. We therefore investigated ISEL in relation to PMD and fixation in rat and human brain. Application on a unilateral lesion model in perfused rat brain revealed that prolonged post-fixation strongly diminished ISEL results. However, microwave pre-treatment can counteract these masking effects without inducing nonspecific labeling contralaterally. On the other hand, in briefly post-fixed, perfused brain or immersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious and induced strong nonspecific labeling. In young rat brain, PMD did not influence the low numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labeling occurred. In human cortex, DNA fragmentation patterns were independent of duration of fixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggest that ISEL on human brain represents antemortem DNA damage rather than PMD artifacts. Furthermore, microwave pre-treatment appears beneficial only in particular fixation conditions.
Use of double labeling and photo CD for morphometric analysis of injured skeletal muscle.
Authors: Stauber W T , Miller G R , Gibala M J , MacDougall J D
Published: April 2011

We used computer-assisted analysis of myofiber cross-sectional areas to measure skeletal muscle responses to injury and disease. We developed a simple, inexpensive method for measuring myofiber size in human muscle samples using Kodak photo compact discs (CDs) as the image source. The photo CD serves as a permanent image storage medium and provides a high-resolution image that can be used to detect small myofibers. The use of double labeling for dystrophin and desmin allowed positive identification of both degenerating and regenerating fibers in a single biopsy specimen.
Multidrug resistance P-glycoprotein monoclonal antibody JSB-1 crossreacts with pyruvate carboxylase.
Authors: Rao V V , Anthony D C , Piwnica-Worms D
Published: April 2011

Multidrug resistance (MDR) is associated with overexpression of a 170 KD plasma membrane P-glycoprotein (P-gp), a putative energy-dependent efflux transporter that reduces intracellular accumulation of chemotherapeutic agents. For detection of P-gp expression in normal and malignant tissues, an MDR1-specific monoclonal antibody (MAb) JSB-1 has been used extensively. In this report we show that MAb JSB-1 crossreacts with a protein of M(r) approximately 130,000 present in rat liver mitochondrial inner membrane/matrix fractions. Peptide mapping and microsequencing identify this protein as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. MAb JSB-1 also crossreacts with purified PC from bovine liver. Under immunoblotting conditions, this crossreactivity is partially abolished by pre-incubation of MAb JSB-1 with a 1000-fold molar excess of MAb C494 epitope-specific peptide (PNTLEGN), indicating that the epitope of MAb JSB-1 may either overlap with or be in close proximity to that of MAb C494. Immunohistochemical cross-reactivity was also demonstrated in cryosections of human skeletal muscle, a tissue known not to express P-gp. MAb JSB-1 strongly immunostained Type 1 fibers, the subtype known to contain abundant mitochondria. Use of MAb JSB-1 for detection of MDR1 P-gp expression should be approached with caution.
Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium.
Authors: Stirling J W , Graff P S
Published: April 2011

To optimize the ultrastructural localization of immunoglobulin G in corneal crystalloid deposits, we compared a range of antigen unmasking techniques. A human corneal biopsy specimen was fixed in formalin, post-osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium ethoxide or sodium metaperiodate. Sections were also treated by heating them at 95 degrees C while they floated on water, 0.01 M citrate buffer (pH 6.0), or sodium metaperiodate. The treatments were applied separately and combined. After labeling, crystalloids in untreated sections had a probe density of 5 particles/microns2. Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe densities of 15-20 particles/microns2. Sodium ethoxide combined with heating on water, or citrate buffer, gave probe densities of 140-160 particles/microns2. Sodium metaperiodate combined with heating on citrate buffer gave the highest probe density (195 particles/microns2). Although sodium ethoxide coupled with heating increased probe density, the ethoxide etched the sections and caused unacceptable damage. Treatment with sodium metaperiodate followed by heating on citrate buffer is recommended for antigen unmasking. This combination gave a high probe density and sections remained intact, with good ultrastructural detail.
Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.
Authors: Mazzotti G , Zini N , Rizzi E , Rizzoli R , Galanzi A , Ognibene A , Santi S , Matteucci A , Martelli A M , Maraldi N M
Published: April 2011

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.
Regional and maturation-associated expression of endothelin 2 in rat gastrointestinal tract.
Authors: de la Monte S M , Quertermous T , Hong C C , Bloch K D
Published: April 2011

Endothelin 2 (ET2), also referred to as vasoactive intestinal contractor peptide, is a member of a family of vasoactive peptides. ET2 is a potent constrictor of intestinal smooth muscle, and the mRNA that encodes it has been detected in murine intestinal extracts. To further investigate the potential physiological roles of ET2, we characterized the cellular distribution of ET2 gene expression in adult rat gastrointestinal tract. Using an RNAse protection assay, an overall proximal to distal gradient of increasing ET2 gene expression was observed from stomach to colon. In situ hybridization studies confirmed this finding and demonstrated ET2 mRNA localized in lamina propria stromal cells. Moreover, ET2 gene expression in stromal cells increased from crypt to villous tip. The results demonstrate that ET2 is produced by stromal cells in villi throughout the intestine. Increased ET2 gene expression at the villous tip is associated with more mature overlying epithelial cells, suggesting a possible role for this vasoactive peptide in intestinal epithelial differentiation or secretory activity.
Lectin-binding properties of human breast cancer cell lines and human milk with particular reference to Helix pomatia agglutinin.
Authors: Schumacher U , Adam E , Brooks S A , Leathem A J
Published: April 2011

Several studies have shown binding of a variety of lectins to breast cancer cells in tissue sections. In particular, binding of the lectin from the Roman snail, Helix pomatia agglutinin (HPA), to breast cancer cells is linked with a poor prognosis. The molecular basis for lectin binding to metastatic breast cancers is not known. To elucidate this in a model system, lectin-binding patterns of seven human breast cancer cell lines were investigated, their cell membranes were isolated, and HPA binding was assessed. In addition, the influence of fixation and processing on lectin-binding sites was also investigated. Binding of lectins to the tumor cells was very heterogeneous between and within the different cell lines and was influenced by fixation and processing. However, some cell lines showed HPA-binding sites both in vivo and in tissue sections. Analysis of the isolated cell membrane glycoproteins from these cell lines on Western blots revealed that HPA can bind to several membrane glycoproteins. In contrast, human milk shows only one major milk glycoprotein that is HPA-positive. Therefore, a switch in glycosylation appears to be taking place during the transformation to a metastatic phenotype.
Microwave fixation improves antigenicity of glutaraldehyde-sensitive antigens while preserving ultrastructural detail.
Authors: Jamur M C , Faraco C D , Lunardi L O , Siraganian R P , Oliver C
Published: April 2011

Microwave fixation for electron microscopy has been used primarily for post-embedding immunocytochemistry. The present study examined the ability of microwave fixation to preserve the antigenicity of glutaraldehyde-sensitive antigens for pre-embedding immunocytochemistry. Five monoclonal antibodies (MAbs) directed against cell surface components of rat mast cells were tested. The MAbs failed to show any labeling of conventionally fixed rat bone marrow-derived mast cells even at glutaraldehyde concentrations as low as 0.1%. Strong staining of mast cell plasma membranes was seen when bone marrow was initially fixed with 2% formaldehyde and then refixed in 2% glutaraldehyde/2% formaldehyde after immunostaining. However, the ultrastructural preservation of the cells was poor. Antigenicity and morphological detail were both preserved when bone marrow was fixed in 0.05% glutaraldehyde/2% formaldehyde for 4 sec in a 550-W microwave oven. With this method, mast cells in various stages of maturation as well as cells that did not contain granules were immunoreactive. This method should prove useful with antigens from many different cell types that are sensitive to glutaraldehyde fixation.
Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.
Authors: Whiteland J L , Nicholls S M , Shimeld C , Easty D L , Williams N A , Hill T J
Published: April 2011

We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.
Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.
Authors: Sun X J , Tolbert L P , Hildebrand J G
Published: April 2011

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.
The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study.
Authors: Roark E F , Keene D R , Haudenschild C C , Godyna S , Little C D , Argraves W S
Published: April 2011

We examined the pattern of fibulin-1 mRNA and protein expression in human tissues and cell lines. Fibulin-1 transcripts were found in RNA isolated from most tissues and a variety of cultured cells, including fibroblasts, smooth muscle cells, and several epithelial cell lines, but not endothelial cells, lymphomyloid cells, or a number of carcinoma and melanoma lines. Immunohistochemical analysis showed that fibulin-1 is an intercellular component of connective tissues, predominantly associated with matrix fibers in tissues such as the cervix, dermis, intimal and medial layers of blood vessels, heart valves, meningeal tissue of the brain, Wharton's jelly of the umbilical cord, testis, and lung. Most of the fibers that were immunoreactive with fibulin-1 antibodies also stained with antibodies to the elastic fiber proteins elastin and fibrillin, as well as with Verhoeff's elastin stain. Immunoelectron microscopic analysis of elastin fibers of skin and saphenous vein revealed that fibulin-1 was located within the amorphous core of the fibers, similar to elastin, but it was not in the fibrillin-containing, elastin-associated microfibrils. Our finding that fibulin-1 is an elastic fiber component suggests several possible new functions for fibulin-1, e.g., that it is a structural protein that contributes to the elastic properties of connective tissue fibers or that is involved with the process of fibrogenesis.
Immunostaining of chain-specific keratins on formalin-fixed, paraffin-embedded tissues: a comparison of various antigen retrieval systems using microwave heating and proteolytic pre-treatments.
Authors: Hazelbag H M , van den Broek L J , van Dorst E B , Offerhaus G J , Fleuren G J , Hogendoorn P C
Published: April 2011

The use of chain-specific monoclonal antibodies (MAbs) against keratins in pathology is hampered by their limited staining on formalin-fixed, paraffin-embedded tissue. In the present study, various treatments before immunohistochemistry on paraffin sections were compared, including proteolytic enzymes and microwave antigen retrieval in various solutions. Sections of normal cervical and skin tissue were stained in a three-step immunoperoxidase method, employing a broad panel of MAbs against chain-specific keratins 4, 5, 7, 8, 10, 13, 14, 17, 18, 19 and pankeratin. Using microwave heating, Target Unmasking Fluid (TUF), Antigen Retrieval Solution (ARS), a simple detergent solution (DET), PBS, and distilled water (MiQ) were compared. Microwave heating in PBS or MiQ strongly improved staining results. Moreover, microwave pre-treatment in TUF or DET gave excellent and specific staining with the majority of MAbs tested, comparable with or even better than staining obtained on frozen sections. Using microwave antigen retrieval, tissue morphology remained optimal, and only in a very limited number of MAbs did immunoreactivity on paraffin sections fail to be restored. Proteolytic pre-treatment with trypsin, pepsin, or pronase gave moderate to strong staining with some of the MAbs. Other MAbs, for which microwave pre-treatment was able to restore the loss of immunoreactivity, failed to give appropriate staining with proteolytic pre-treatment. Our results show that microwave heating in either TUF or a simple detergent solution before immunohistochemistry is a reliable method for antigen retrieval of chain-specific keratins in formalin-fixed, paraffin-embedded tissues.