bluuebunny/crossref_metadata_2025 huggingface dataset:
Transforming growth factor-alpha immunoreactivity during induced hair follicle growth cycles in sheep and ferrets.
Authors: Nixon A J , Broad L , Saywell D P , Pearson A J
Published: April 2011

Transforming growth factor-alpha (TGF-alpha) has been associated with cell proliferation of keratinocytes and implicated in hair growth. We therefore examined changes in the immunocytochemical localization of TGF-alpha and cell proliferation markers in the skin of two unrelated species in which hair cycles could be induced, to elucidate the role of this growth factor in the control of fiber growth. Skin was collected from melatonin-treated ferrets (Mustela putorius furo), untreated Romney sheep (Ovis aries), and New Zealand Wiltshire sheep in which interruption of wool growth had been photoperiodically induced. Immunostaining patterns were very similar in ferrets and sheep. TGF-alpha immunoreactivity was observed in epithelial tissues of the skin but was not co-localized with cell proliferation markers. In anagen follicles, specific staining was most intense in the innermost cells of the outer root sheath and cortical cells in the keratogenous zone but was absent from inner root sheath or dermal papilla. TGF-alpha immunostaining diminished during catagen, although faint staining was retained in all epithelial cells. In telogen and early proanagen follicles, staining remained faint or was restricted to cells on the margin of the brush end and follicle neck. Immunoreactivity in the outer root sheath was reestablished in late proanagen. Sebaceous glands and epidermis were stained intensely throughout the hair cycle. TGF-alpha-immunoreactive components of skin extracts, analyzed by Western blotting, showed mobility corresponding to approximately 32 KD, but not to the size of the fully cleaved peptide. These results are consistent with an epithelial autocrine or juxtacrine, but not a mitogenic, function of TGF-alpha.
Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol.
Authors: van Gijlswijk R P , Wiegant J , Raap A K , Tanke H J
Published: April 2011

Recently, a peroxidase-mediated amplification system has been described for immunofluorescence and fluorescence in situ hybridization studies. It is based on the deposition of hapten- or fluorochrome-labeled tyramide molecules. Although providing a significantly increased detection sensitivity compared to conventional procedures, its localization properties are inferior because of free diffusion of intermediate reaction products before they are immobilized. In enzyme cytochemistry, it is well established that improved localization of enzyme activity can be achieved through the addition of viscosity-increasing polymers to the incubation media. In this study we analyzed the effect of different polymers on the localization sharpness and sensitivity of the tyramide-peroxidase reaction in FISH applications. Significantly improved localization of the fluorescent endproduct was observed using dextran sulfate or polyvinylalcohol (PVA) with, respectively, no or little loss of sensitivity.
A method for staining of cell nuclei in Xenopus laevis embryos with cyanine dyes for whole-mount confocal laser scanning microscopy.
Authors: de Mazière A M , Hage W J , Ubbels G A
Published: April 2011

To study the cell cleavage pattern in experimentally treated Xenopus laevis blastulae, we devised a method to visualize all cell nuclei, whether in interphase or in a mitotic phase, in whole-mount embryos using confocal laser scanning microscopy. Optimal staining conditions were defined for the recently commercialized cyanine nucleic acid stain TO-PRO-3, which is excited by a 647-nm laser beam and fluoresces in the far red of the spectrum. This is beyond the spectral range of autofluorescence caused by most biomolecules and, in particular, by the high amount of yolk granules in these embryos. The quality of the TO-PRO-3 image was compared to that after nuclear staining with BOBO-3, another cyanine dye that fluoresces at slightly shorter wavelengths. In the proposed procedure, special attention is paid to permeabilization of the membranes to the dyes and to bleaching of the natural pigment of the embryos with maximal preservation of cellular and nuclear structures. Because of its emission maximum at 661 nm, TO-PRO-3 is a promising nuclear stain for specimens with special background problems and for multicolor fluorescence microscopy.
Ultrastructural localization of the C-propeptide released from type II procollagen in fetal bovine growth plate cartilage.
Authors: Lee E R , Smith C E , Poole R
Published: April 2011

We used immunochemical and immunoelectron gold techniques to determine whether the C-propeptide previously identified in the matrix of endochondral cartilage (CPII) was still a part of the Type 11 procollagen molecule or had been released from it. Guanidinium hydrochloride extraction, followed by SDS-PAGE and Western blotting techniques and immunoelectron localization, revealed that predominantly only the released form (hereafter referred to as released CPII) was detected. The ultrastructural distribution of this CPII was examined with affinity-purified antibodies and with immunogold or immunoperoxidase localization techniques in the presence or absence of embedding resins. These methods yielded similar results. Although no significant amount of this CPII was retained in the matrix after guanidinium hydrochloride extraction, it was present in two recognizable sites under normal conditions, i.e., locally concentrated in a random association with collagen fibrils in the nonmineralized matrix and mainly concentrated in interfibrillar mineralizing sites in the mineralized matrix. These results suggest that the C-propeptide that has been released from Type II procollagen associates with collagen fibrils and then preferentially associates with mineralizing sites when these form in the endochondral cartilage. The significance of this preference for mineral is not known but may have something to do with its high affinity for hydroxyapatite.
Localization of endothelial NOS at the basal microtubule membrane in ciliated epithelium of rat lung.
Authors: Xue C , Botkin S J , Johns R A
Published: April 2011

Nitric oxide (NO), an important cell messenger molecule, is formed endogenously in the lung airway. Three individual genes of NO synthase (NOS), which represent brain NOS (bNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), have been reported in the cultured lung epithelium. Although studies in vivo showed that bNOS and iNOS were expressed and localized in the cytoplasm of bronchial epithelium, the expression and localization of eNOS remains to be determined. Therefore, we employed an eNOS monoclonal antibody whose immunospecificity was tested by both Western blot and preadsorption immunohistochemistry to immunostain rat lungs from fetus to adult. The results showed that eNOS immunoreactivity began to appear in the lung epithelium within 2 hr after birth. Six hours later (8 hr after birth), the NOS immunoreaction was concentrated near the surface of the ciliated epithelial cells. This staining pattern appeared in lungs at Day 1, Week 1, Week 2, and in adult rats. By electron microscopy, eNOS immunoreactivity was confirmed within ciliated epithelium and was shown to be associated with the basal microtubule membrane of the cilia. Nonciliated cells were not stained. Type II epithelial cells also contain eNOS immunoreactivity, which is primarily associated with rough endoplasmic reticulum, and free ribosomes. However, macrophages in the lungs lacked eNOS immunoreactivity. This study demonstrated that eNOS was postnatally expressed in rat bronchial ciliated epithelium. The localization of eNOS at the basal membrane of ciliary microtubules suggests that eNOS may be involved in the function of epithelial cilia, consistent with previous physiological studies.
Development of GP-2 and five zymogens in the fetal and young pig: biochemical and immunocytochemical evidence of an atypical zymogen granule composition in the fetus.
Authors: Lainé J , Pelletier G , Grondin G , Peng M , LeBel D
Published: April 2011

To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.
An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization.
Authors: Wiegant J , Verwoerd N , Mascheretti S , Bolk M , Tanke H J , Raap A K
Published: April 2011

Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.
Semi-automated positional analysis using laser scanning microscopy of cells transfected in a regenerating newt limb.
Authors: Pecorino L T , Brockes J P , Entwistle A
Published: April 2011

Limb regeneration in urodele amphibians such as the newt is a key system for investigating the positional identity of cells. The regenerate arises locally from blastemal cells, mesenchymal progenitors that normally give rise to structures distal to the amputation plane but which can be respecified (proximalized) by treatment with retinoic acid (RA) such that proximal structures are formed. To establish an assay for positional identity, cells of distal and RA-treated distal blastemas are labeled by transfection with an alkaline phosphatase marker gene using particle bombardment (biolistics). After grafting the distal blastema to a proximal stump, a context known as intercalary regeneration, the proximodistal distribution of labeled cells in the resulting regenerate is an index of positional identity. We use enzyme-labeled fluorescence (ELF) in conjunction with laser scanning microscopy to detect transfected cells within a section of the entire regenerate. A semi-automated analysis of the positional distribution of marked cells along the proximal-distal axis demonstrates that cells from both distal and RA-treated blastemas contribute to the regenerate. This procedure provides an efficient and accurate tool for positional analysis of transfected cells, and should be applicable for studying genes that play a role in specifying cell position during morphogenesis.
Use of serial semithin frozen sections to evaluate the co-localization of estrogen receptors and progesterone receptors in cells of breast cancer tissues.
Authors: Kudo A , Fukushima H , Kawakami H , Matsuda M , Goya T , Hirano H
Published: April 2011

The extent of co-expression of estrogen receptors (ER) and progesterone receptors (PgR) in breast cancer cells was examined immunocytochemically. Eight surgical cases of infiltrating ductal carcinoma designated as ER-positive and PgR-positive (ER+/PgR+) by enzyme immunoassay (EIA) were used. They were fixed with 4% formaldehyde and cut into serial frozen semithin sections. Using sections stained with either anti-ER or anti-PgR antibody, we ascertained the co-localization of ER and PgR in a single cell and estimated the ratio of the number of cells co-expressing ER and PgR. Twenty-six to 95% of the cells were immunopositive for both ER and PgR, 2-25% of them, varying in cases, were positive for ER but not for PgR, and <3% of the cells were positive for PgR but not for ER. The remaining 5-60% cells were positive for neither ER nor PgR. A significant percentage of breast cancer cells in tissues designated as ER+/PgR+ by EIA showed the phenotype of ER-positive but PgR-negative. The co-expression ratio of ER and PgR in biochemically detected ER+/PgR+ breast cancer may reflect a particular clinical parameter, such as the heterogeneous responsiveness of ER+/PgR+ breast cancers to hormonal treatment. Immunostaining of serial semithin frozen sections for two or more different antigens is a useful method to assess the correlation of localization of antigens.
High-efficiency expression gene cloning by flow cytometry.
Authors: Dell'Arciprete R , Stella M , Fornaro M , Ciccocioppo R , Capri M G , Naglieri A M , Alberti S
Published: April 2011

Our goal was to develop a convenient and widely applicable procedure for gene cloning based on flow cytometry. To this purpose, we have developed an efficient protocol for DNA transfection and selection of rare transfectants. Transfection by calcium phosphate co-precipitation was extensively investigated. The use of specific batches of calcium chloride, of carrier DNA purified in guanidinium thiocyanate, and of plasmid DNA banded in cesium chloride proved crucial for high efficiency of transfection. Several tissue culture parameters were also found critical. With the optimized procedure we can transfect almost 100% of the COS-7 cells with cDNA encoding cell surface antigens or green fluorescent protein. Moreover, we routinely obtain high average levels of expression. Efficient cell sorting in flow cytometry was achieved by subtracting the cell autofluorescence background, by displacing stained cells in the red dimension, and by combining fluorescein-conjugated primary and secondary antibodies. Efficient recovery of the transfected DNA constructs was obtained from 2500-3000 cells directly sorted in Hirt lysis buffer. Using the above protocol we have cloned by expression the gene encoding Trop-2, a cell surface glycoprotein expressed by human carcinomas.
Bifunctional protein cross-linking reagents improve labeling of cytoskeletal proteins for qualitative and quantitative fluorescence microscopy.
Authors: Safiejko-Mroczka B , Bell P B
Published: April 2011

Because permeabilization of the cell membrane is necessary to label intracellular proteins with most fluorescent probes, it is important to optimize the preservation and labeling of the proteins under study. We used qualitative and quantitative fluorescence microscopy to evaluate the effects of six different bifunctional protein cross-linking reagents and several extraction conditions on the labeling of filamentous actin with phalloidin and the immunolabeling of tubulin and gelsolin. The labeling of cytoskeletal and associated proteins can be significantly enhanced by the appropriate combination of bifunctional protein cross-linking reagents and extraction conditions. However, the conditions that give the most intense labeling vary depending on the label used. The greatest intensity of labeling with either phalloidin or antibodies was obtained with the intermediate-length cross-linker DSP. The two-step procedure of cross-linking with DSP and extracting in Triton X-100 in microtubule-stabilizing buffer containing DSP gives maximal labeling with phalloidin. Maximal labeling of gelsolin and tubulin with antibodies is obtained by extracting DSP-cross-linked cells with Triton in Hank's saline containing DSP. Therefore, DSP reproducibly improves preservation of both soluble and filamentous proteins for quantitative and qualitative studies by fluorescence microscopy.
Cellular localization of P-glycoprotein in brain versus gonadal capillaries.
Authors: Stewart P A , Béliveau R , Rogers K A
Published: April 2011

P-glycoprotein, the multidrug resistance protein that actively transports a wide variety of lipophilic substrates out of cancer cells, has recently been described in some normal tissues, including the endothelium of the brain and testes. Here we show that P-glycoprotein is also expressed in ovarian endothelium. In ovarian capillaries, the immunolabeled protein was detected with two monoclonal antibodies to P-glycoprotein. It was shown to be membrane-bound and to transport a known P-glycoprotein substrate. Expression of P-glycoprotein in endothelial cells suggests that this transport protein plays a role in enhancing or restricting vascular permeability to lipophilic molecules. If it does, then its role may be predicted from its site of expression on the luminal or abluminal face of the capillary wall. In the region of the endothelial nucleus, endothelial membranes are sufficiently far apart that they can be distinguished at the light microscopic level. Confocal examination of tissue sections double labeled for P-glycoprotein and nuclei confirmed that, in brain, P-glycoprotein is expressed only on luminal membranes. This location is consistent with its putative role in protecting the neuropil from circulating lipophilic molecules. In both testicular and ovarian endothelium, however, P-glycoprotein is expressed on both luminal and abluminal membranes. This localization suggests that it acts to exclude P-glycoprotein substrates from the endothelial cells themselves.
Internalization of styryl dye FM1-43 in the hair cells of lateral line organs in Xenopus larvae.
Authors: Nishikawa S , Sasaki F
Published: April 2011

We used a fluorescent dye, FM1-43 to investigate mechanotransduction mechanisms in the hair cells of lateral line organs of Xenopus larvae. FM1-43 specifically labeled the hair cells. The photo-oxidation technique was performed with election microscopy to examine the labeling sites and their mechanisms. The results showed that the labeling sites were mitochondria and rough endoplasmic reticulum throughout the cytoplasm. Endocytic activity of the hair cells was limited to endosomes and small granules located at the apical part of the cells. Blockers of the mechanosensitive cation channel (neomycin, gentamicin, streptomycin, and amiloride) effectively inhibited FM1-43 labeling. One of the blockers, amiloride, was found to bind to hair cells when its fluorescence was examined. Divalent cations such as Mg2+ and Ca2+, but not monovalent cations such as Na+ and K+, inhibited FM1-43 labeling when they were added in excess amounts. These results suggest that FM1-43 internalizes hair cells via the putative mechanosensitive cation channel in the plasma membrane.
Possible association of chaperonin 60 with secretory proteins in pancreatic acinar cells.
Authors: Le Gall I M , Bendayan M
Published: April 2011

Assembly and folding of newly synthesized polypeptides, acquisition of their biological active form, and their translocation in different cellular compartments are processes assisted by molecular chaperones. Because particular chaperones have been found to be present along the RER-Golgi-granule secretory pathway in pancreatic acinar cells, we presume that they should play important roles in secretion. In the present study, applying double immunogold labeling at the electron microscopic level on rat exocrine pancreas, we have revealed the existence of a topographical association between Hsp60 and particular pancreatic enzymes along the secretory pathway. The highest association was found for amylase, lipase, and chymotrypsinogen, whereas trypsinogen and carboxypeptidase B showed much lower association values. Immunoprecipitation of isolated zymogen granule content with an anti-Hsp60 antibody appears to confirm the morphological data, since amylase and lipase were found to co-precipitate with Hsp60. These findings support the hypothesis that Hsp60 is associated with certain pancreatic proteins along the secretory pathway. Hsp60 would assist the proper folding and assembly of pancreatic secretory proteins and could also prevent their autoactivation before secretion.
Immunohistochemical detection of thrombospondin-1 in formalin-fixed, paraffin-embedded tissue.
Authors: Grossfeld G D , Shi S R , Ginsberg D A , Rich K A , Skinner D G , Taylor C R , Cote R J
Published: April 2011

Thrombospondin-1 (TSP) is a 450-KD glycoprotein that was initially discovered in the platelet alpha-granule. It now appears that TSP is intimately involved in the regulation of a variety of cellular functions and cell-to-cell interactions. Recently, it has been demonstrated that TSP functions as a p53-dependent inhibitor of angiogenesis in cultured fibroblasts from Li-Fraumeni patients and therefore may be an important factor involved with tumor invasion and metastasis. It has previously been demonstrated that TSP can be detected in frozen tissue sections by immunohistochemical methods. Our objective in this study was to determine the optimal antigen retrieval (AR) protocol for detection of TSP in formalin-fixed, paraffin-embedded tissue by using tissue sections from patients with invasive transitional cell carcinoma of the bladder. The optimal AR protocol was determined utilizing a variety of heating conditions and antigen retrieval buffers. Our results demonstrate that TSP can be reliably detected in paraffin-embedded tissue by immunohistochemical techniques that utilize AR with high-temperature microwave heating and a low-pH Tris-HCI buffer. The importance of this method is that it allows the reliable detection of TSP in archival tissue. This should facilitate further investigation into TSP's role in the regulation of cellular processes, including its influence on tumor angiogenesis and metastasis.
PYY in developing murine islet cells: comparisons to development of islet hormones, NPY, and BrdU incorporation.
Authors: Jackerott M , Oster A , Larsson L I
Published: April 2011

Exhaustive characterizations of antisera to the structurally related peptides pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY) enabled us to establish the developmental pattern of these peptides in rat and mouse pancreas. PYY was the earliest detectable peptide and was present in all early appearing endocrine cell types. NPY appeared later and occurred exclusively in a subpopulation of insulin cells, whereas PP cells arose latest. At the earliest stage studied, all endocrine cells stored PYY. Most of these cells also contained glucagon. Subsequently, the endocrine cells comprised glucagon+PYY cells and glucagon+PYY+insulin cells. Later, cells storing either only insulin or insulin+PYY appeared. Quantitations of the relative numbers of these cell populations during development were consistent with a precursor role of triple-positive (insulin+glucagon+PYY) cells. Moreover, bromodeoxyuridine (BrdU) injections at E15.5 showed that a large percentage of triple-positive cells were in S-phase and therefore were actively dividing, whereas almost no pure insulin cells or insulin+PYY cells synthesized DNA at this time. These results suggest that PYY-positive endocrine cells may represent precursors for mature islet cells.
Interspecies reactivities of anti-human macrophage monoclonal antibodies to various animal species.
Authors: Zeng L , Takeya M , Ling X , Nagasaki A , Takahashi K
Published: April 2011

We examined interspecies reactivities of eight anti-human monocyte/macrophage monoclonal antibodies (MAbs), Am-3K, PM-2K, X4, X14, Ber-MAC3, GHI/61, EBM/11, and KP1, with various animal tissues including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovines, horses, and monkeys. All MAbs recognized monkey macrophages. Pig macrophages were detected by most MAbs except for EBM/11 and KP1. Of the eight antibodies, AM-3K showed the widest interspecies reactivity. It reacted with macrophages of all animal species examined, except for rats. Western blot analysis revealed a similarity in the antigens recognized by AM-3K among guinea pigs, rabbits, and humans. Other anti-human MAbs demonstrated distinct reactive patterns against macrophages in animals. The immunostaining patterns of all of these MAbs in animal tissues were similar to those found in humans, although some MAbs, such as AM-3K, EBM/11, and X4, displayed more restricted reactivity in animals than in humans. These results indicate that some anti-human monocyte/macrophage MAbs are also available for immunohistochemical detection of monocyte/macrophages in animal tissues. Among them, AM-3K is considered to be the most useful MAb for identifying macrophages in various tissues of animals.
Direct visualization of copper-metallothionein in LEC rat kidneys: application of autofluorescence signal of copper-thiolate cluster.
Authors: Okabe M , Nakayama K , Kurasaki M , Yamasaki F , Aoyagi K , Yamanoshita O , Sato S , Okui T , Ohyama T , Kasai N
Published: April 2011

We report on the histochemistry of copper-metallothionein (Cu-MT) in the kidneys of Long Evans Cinnamon (LEC) rats. We used the visualization principle of histochemistry based on the autofluorescence emission from the fluorophore of Cu(+)-thiolate clusters in proteins. Intense autofluorescence signals were observed with a ring at the outer stripe of the outer medulla. Orange fluorescence signals were observed in the nuclei and cytoplasm of proximal straight tubular (PST) cells of segment 3 (S3) at the outer stripe of the outer medulla, and yellow-orange signals were detected in lysosome-like organelles in the proximal convoluted tubule (PCT) cells of segments 1 and 2 (S1 and S2) adjacent to the glomeruli in the cortex. These fluorescent materials were identified as Cu-MT because both signals were quenched by withdrawing Cu+ or by blocking cysteine residues, the distributions of cysteine residues and immunoreactive MT showed identical patterns to the localization of the fluorescence signals, and the fluorescent proteins containing Cu were eluted at the same Kd value of purified Cu-MT by gel filtration chromatography. However, a high level of MT mRNA was detected only in the outer stripe of the outer medulla where the orange fluorescence signals were detected, but not in the cortex. This difference in localization between the protein and the mRNA suggested that synthesis of renal MT occurs do novo in the outer stripe of the outer medulla. The yellow-orange fluorescent Cu-MT located in the lysosomal organelles at S1 and S2 of the PCT cells in the cortex could be Cu-MT of nonrenal origin, i.e., Cu-MT transported from other organs.
Transient expression of collagen type XIV during muscle development and its reappearance after denervation and degeneration.
Authors: Tono-Oka S , Tanase S , Miike T , Tanaka H
Published: April 2011

In the formation of muscle pattern, the architectural arrangement is believed to be controlled by the local connective tissue cells. In this study we examined the immunohistological localization of Type XIV collagen recognized by a monoclonal antibody, MAb DBM, in embryonic chick hind limbs from stage (St.) 27 to 2 weeks post hatching. DBM staining was transiently observed in the epimysium from St. 30, in the perimysium of the dorsal region from St. 37, and in the entire perimysium from St. 39. After hatching, DBM staining was notably diminished in both epimysium and perimysium. In contrast, DBM staining and in situ hybridization signals for Type XIV collagen mRNA increased in the muscle connective tissues after denervation and around the regenerating muscle fibers. Therefore, Type XIV collagen expression appears to coincide with muscle activity and muscle regenerating conditions, and Type XIV collagen is considered to play roles in muscle development and regeneration.
Islet morphogenesis and stem cell markers in rat pancreas.
Authors: Bouwens L , De Blay E
Published: April 2011

During embryonic development, and possibly also later in life, pancreatic islets of Langerhans originate from differentiating epithelial stem cells. These stem cells are situated in the pancreatic ducts but are otherwise poorly characterized. We found by immunohistochemical staining that protodifferentiated pancreatic epithelial cells from rat embryos of Day 13-Day 15 express the cytoskeletal protein keratin 20, similar to mature duct epithelium. During the period of islet morphogenesis, which occurs between Day 17 and birth, large aggregates of K20-positive duct cells were formed, which gradually differentiated into endocrine cells. This islet morphogenic mechanism has not been described thus far and we did not observe it in postnatal rats. During fetal islet formation, transient expression of vimentin was noted in the duct cells but not in endocrine cells. This intermediate filament protein is not observed in duct epithelial cells after birth. The proto-oncogene product bcl-2, a putative epithelial stem cell marker, was detected in duct cells from fetal and postnatal pancreas. We conclude that K20, vimentin, and bcl-2 are markets for the pancreatic (islet) stem cells.
Transient accumulation of perisinusoidal chondroitin sulfate proteoglycans during liver regeneration and development.
Authors: Yada T , Koide N , Kimata K
Published: April 2011

After partial hepatectomy, the liver is capable of complete restoration of normal hepatic size, architecture, and function (regeneration). To study roles of the extracellular matrix in regeneration, the temporal and spatial sequences of deposition of several components, including collagen I, III, and IV, fibronectin, laminin, heparan sulfate proteoglycan (perlecan), and chondroitin sulfate proteoglycans were characterized by light microscopic immunohistochemistry in rat liver after 70% partial hepatectomy. Consistent with previous reports, there was a brisk mitosis of hepatocytes after the partial hepatectomy. Of the extracellular matrix components studied, 1B5 epitope generated by chondroitinase ABC digestion on chondroitin sulfate proteoglycans exhibited the most dramatic changes; the epitope was detectable as early as 1.5 hr after partial hepatectomy and its immunoreactivity reached a maximum at 24 hr, then declined gradually. This transient expression of the 1B5 epitope was also detected in neonatal rat liver during development. By Western blotting, the 1B5 epitope was found on two forms of the core protein of chondroitin sulfate proteoglycans with apparent molecular masses of 163 KD and 152 KD, which were also regulated in the same temporal manner.
Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study.
Authors: Zaar K
Published: April 2011

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.
M-cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membranes.
Authors: Gebert A
Published: April 2011

The tonsil crypt epithelium contains membranous (M)-cells that transport antigens from the lumen to underlying lymphoid cells, thereby initiating specific immune responses. Mechanisms mediating the adhesion of antigens to the M-cell surface are important for effective and selective uptake of potential pathogens but are still poorly understood. Therefore, the carbohydrates present on crypt epithelial cells of the rabbit palatine tonsil were studied by lectin histochemistry. Ultrathin LR White sections were incubated with a panel of eight lectins conjugated to colloidal gold or biotin. The glycocalyx of the apical membrane of M-cells was selectively labeled by UEA-I, LTA, HPA, and VVA, whereas that of the remaining squamous epithelial cells preferentially bound RCA-I and PNA. WGA and ConA showed only little binding, with no discernible preference for any of the cell types. Double labeling of UEA-1 together with anti-vimentin antibodies revealed that UEA-I-positive epithelial cells also contained the rabbit M-cell marker vimentin, and vice versa. The results show that a specific composition of glycoconjugates, which differs from that on squamous epithelial cells, is found on M-cells of the rabbit tonsil. The M-cell-specific glycoproteins and glycolipids could be selectively targeted by microorganisms that adhere to M-cells and enter the host along this pathway.
Optimization of immunofluorescence methods by quantitative image analysis.
Authors: Mosedale D E , Metcalfe J C , Grainger D J
Published: April 2011

There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.
Cellular and subcellular localization of acetaldehyde-protein adducts in liver biopsies from alcoholic patients.
Authors: Paradis V , Scoazec J Y , Köllinger M , Holstege A , Moreau A , Feldmann G , Bedossa P
Published: April 2011

Acetaldehyde, the first product of ethanol in hepatocytes, can react with protein to form acetaldehyde-protein adducts (APAs). Because it has been suggested that these adducts could be involved in the pathogenesis of ethanol-induced hepatic lesions and in fibrogenesis, we performed an ultrastructural immunohistochemical study to precisely define the cellular and subcellular localization of APAs. A preembedding technique of indirect immunoperoxidase was performed in liver biopsy specimens from eight patients with alcoholic liver disease, using a specific antiserum against APAs. In all specimens, APAs were detected in the rough endoplasmic reticulum, in some peroxisomes, and in the cytosol of hepatocytes. In four patients with steatofibrosis or cirrhosis, labeling of Ito cells was also observed. In these cases, the same staining pattern was observed in the cytoplasmic processes of myofibroblasts in areas of fibrogenesis. When isolated rat Ito cells were incubated in the presence of acetaldehyde, APAs were also detected in the cytoplasm. These results show that APA formation occurs in hepatocytes at the sites of acetaldehyde production. Detection of APAs in human and rat Ito cells strongly suggests that acetaldehyde can diffuse into Ito cells and bind to cytoplasmic proteins to form local APAs. Because Ito cells are the main effector cells of liver fibrosis, detection of APAs in these cells points to their possible involvement in liver fibrogenesis.
Use of PG-21 immunocytochemistry to detect androgen receptors in the songbird brain.
Authors: Smith G T , Brenowitz E A , Prins G S
Published: April 2011

The avian song control system is an excellent model in which to study the effects of gonadal steroid hormones on neural and behavioral plasticity. Several of the brain regions that control song behavior concentrate androgens and/or estrogens. Investigations of the distribution and regulation of androgen receptors have been limited by the lack of a reliable immunocytochemical method to detect androgen receptors in the songbird brain. We describe a protocol by which the PG-21 polygonal antibody to the rat androgen receptor can be used to label androgen receptor-containing cells in the songbird brain. By treating songbirds of several species with testosterone 90 min before sacrifice and by using relatively low concentrations (0.5 0.75 microg/ml) of PG-21 antibody to reduce nonspecific background staining, we were able to obtain strong specific labeling of cell nuclei in androgen-sensitive brain regions. This technique will facilitate the study of the role of androgens in mediating neural plasticity in the avian brain. Testosterone pretreatment may also facilitate the use of this antibody to label androgen receptors in tissues from a wide array of nonmammalian species.
THE STAINING OF COLLAGEN WITH ELASTIC TISSUE STAINS
Authors: FULLMER HAROLD M. , LILLIE R. D.
Published: April 2011

We have demonstrated that acetylation or benzoylation of formalin-fixed collagen from the human, rat, or mouse induces its reactivity with the elastic tissue stains: orcein, Weigert's resorcin fuchsin, orcinol-new fuchsin, and Verhoeff's, and blocks the Masson trichrome and the Van Gieson stains which are used to identify collagen. Deamination of collagen induces its reactivity with Gomori's aldehyde fuchsin perhaps because of a net shift of the reaction of collagen to the acid side.
THE DIFFERENCE IN PERMEABILITY OF CARTILAGE TO CATIONIC AND ANIONIC DYES
Authors: KANTOR THOMAS G. , SCHUBERT MANWELL
Published: April 2011

It has been shown that fresh cartilage cubes are readily permeable to all of thirteen cationic dyes and one neutral dye tried. They are impermeable to thirteen of fourteen anionic dyes tried. This selective permeability of fresh cartilage to dyes can be altered by treatment which destroys the fixed negative charge of the cartilage. After this treatment, the cartilage cubes are readily permeable to anionic dyes.

It is suggested that this membrane selectivity of cartilage may have physiological meaning.
THE ALKALINE PHOSPHOMONOESTERASE ACTIVITY OF CELL NUCLEI: ACTIVATION BY 0.16M MAGNESIUM
Authors: FEIGIN IRWIN , WOLF ABNER
Published: April 2011

I. The presence of 0.16M magnesium chloride in the incubation mixture for the Gomori method for localizing alkaline phosphatase activity, markedly increases the enzyme activity in the nuclei of the guinea pig endometrium, ovary and adrenal cortex, rendering them visible after 5 minutes' incubation. The activity in cytoplasmic and extracellular sites is decreased. The staining of these same nuclei could be demonstrated by the Loveless and Danielli method of visualizing the phenolic component of a complex phosphate ester instead of precipitating the phosphate ions with calcium salts. The activity described may be specific for the tissues mentioned and serves as an instance in which nuclear staining in phosphatase preparations is clearly valid.

II. The effect of magnesium and cyanide ions and other factors on the enzyme activity noted histochemically in nuclei and biochemically in nuclear cell fractions, was reviewed. The data support the validity of some earlier histochemical observations describing a nuclear phosphomonoesterase activity in many organs which differs from that in cytoplasmic and extracellular sites. They also suggest that the phosphatase activity in cell fractions of those biochemical preparations prepared in aqueous media are altered by diffusion and adsorption phenomena.
THE SPECIFICITY OF THE PERIODIC ACID-SCHIFF TECHNIQUE STUDIED BY A QUANTITATIVE TEST-TUBE METHOD
Authors: HOOGHWINKEL G. J. M. , SMITS G.
Published: April 2011

A method is described for the quantitative estimation of periodic acid-Schiff positive substances in solutions of tissue components.

It was found that hyaluronic acid and chondroitinsulphuric acid are periodic acid-Schiff negative.

Any coloration observed could be ascribed to contaminating polysaccharides probably not or not exclusively composed of hexosamine and hexuronic acid. Unlike the hexosamine groups, the hexuronic acid groups in hyaluronic acid and chondroitinsulphuric acid are attacked by periodic acid without formation, however, of aldehyde groups.

The chemistry of this process is discussed.
CYTOCHEMISTRY OF PINEAL LIPIDS IN RAT AND MAN
Authors: QUAY W. B. , ABRAMSON CONNIE
Published: April 2011

Lipid cytochemistry studied in 130 rat pineals demonstrates two common types of parenchymal cells which may be involved in an endocrine secretory activity. The first cell type is characterized by its content of abundant lipid droplets, composed primarily of ethanol-soluble carbonyl lipids coated with phospholipid. The second cell type is distinguished by its content of a phospholipid cytoplasmic matrix containing few droplets on vacuoles. This cell type appears to be identical with the cell staining with orange G or phloxine in a modified chrome alum hematoxylin technique described previously.

Parenchymal cells containing lipid occur in seven human pineals of diverse ages; and in the older pineals interlobular cells containing lipid occur as well. The lipid in human parenchymal cells differs from that in the type 1 parenchymal cells of the rat by, (1) forming smaller droplets, (2) containing a greater amount of neutral lipid and less carbonyl lipid, and (3) being associated with pigment fomation in adults and older people.
A HISTOCHEMICAL STUDY OF THE SHELL GLAND OF SCYLIORHINUS CANICULUS
Authors: THREADGOLD L. T.
Published: April 2011

The shell gland of Scyliorhinus caniculus has been investigated histochemically and chromatographically. On the basis of histochemical reactions the gland is divided into five distinct regions, Zones A to E. These zones secrete respectively a carbohydrate, a carbohydrate substance which is metachromatic, a polyphenol oxidase, a protein, a phenol, and perhaps a phenolic protein and a basic protein.

With reference to the histochemical findings and incubation experiments various modes of sclerotin formation are discussed. On the basis of histochemical evidence it is concluded that sclerotin formation could be as deduced by Brunet and Kent (1956) from their work on the oötheca of Blatta and Periplaneta but this is not supported by the incubation experiments. The weight of evidence presented here therefore favours an auto quinone tanning system in the formation of the egg case of Scyliorhinus caniculus.
HISTOCHEMICAL ALDEHYDE BLOCKADE BY ANILINE IN GLACIAL ACETIC ACID
Authors: LILLIE R. D. , GLENNER G. G.
Published: April 2011

More rapid and more complete blockade of tissue aldehydes produced by hydrochloric, periodic or peracetic acid may be achieved by use of molar solutions of aniline in glacial acetic acid than by use of molar aniline hydrochloride in water. A solution of 1% benzidine or o-dianisidine in glacial acetic acid is more effective than the above aniline solution against aldehydes produced by peracetic acid from lipids, but less effective against aldehydes produced by periodic acid from carbohydrates.

Inclusion of 25 parts concentrated hydrochloric acid to 75 parts glacial acetic acid in the solvent impairs or inhibits the azomethine reaction of aniline with tissue aldehydes.

A 20 to 30 minute exposure to a 10% volume dilution of aniline in glacial acetic acid should be adequate for testing the aldehyde nature of Schiff reactive loci in tissue, and in most instances failure of a 3 hour treatment to impair or prevent the following Schiff reaction can be taken to exclude aldehyde.

The greater relative effect of o-dianisidine and benzidine in preventing the Schiff reaction of periodic or peracetic acid oxidized lipoids is of interest and merits further exploration.

The colors produced by the o-dianisidine condensation with tissue aldehydes are disappointingly pale and this reaction would not appear to have any great histologic value.
AN IMPROVED HISTOCHEMICAL METHOD FOR THE DEMONSTRATION OF POTASSIUM
Authors: CROUT J. RICHARD , JENNINGS ROBERT B.
Published: April 2011

1) A technique is described for the histochemical demonstration of potassium using frozen-dried tissue and sodium cobaltinitrite precipitation.

2) Quantitative data show that the method precipitates tissue potassium completely.

3) Several observations are discussed which indicate that no potassium loss or shift occurs during the staining process.
DETERMINATION OF PROTEIN AND LIPID LOST DURING OSMIC ACID FIXATION OF TISSUES AND CELLULAR PARTICULATES
Authors: DALLAM R. DUNCAN , TELFER RUTH CURTISS
Published: April 2011

Heart and kidney fragments and liver mitochondria and microsomes were prepared for electron microscopy by standard methods. The fixative, dehydration and embedding mediums each were analyzed separately for protein and lipid removed. It was demonstrated that protein and lipid were lost from mitochondria and microsomes and that protein was lost from tissue fragments during their preparation for electron microscopic examination. The significance of these results with respect to investigations concerned with the correlation of function to morphology is discussed.