Double immunohistochemical labeling technique applied to different types of cytokeratins in epithelial proliferations of the breast

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.     

A new scoring system using multiple immunohistochemical markers for diagnosis of uterine smooth muscle tumors

The diagnosis of uterine smooth muscle neoplasms by light microscopy is difficult. Multiple classification schemes have been proposed based on mitotic rate, nuclear atypia, and the presence or absence of necrosis. None of these classification systems has been entirely successful. This study was undertaken to evaluate the use of selected immunohistochemical and histochemical markers in differentiating these tumors, in addition to accepted morphologic criteria. Ten cases of each of the following: leiomyosarcomas (LMS), atypical leiomyomas (AL), cellular leiomyomas (CL) and usual leiomyomas (UL), were classically evaluated for histological diagnosis and were stained for Ki-67 (MIB-1), bcl- 2 and p53 using monoclonal antibodies and the avidin-biotin peroxidase method, and argyrophilic nucleolar organizer region (AgNORs). The number of stained cells was counted in the most positively stained region in a 4 mm2 square cover glass mounted on each slide. The mean value was calculated for each group of tumors. The data for Ki-67 (MIB- 1), bcl-2, p53 and AgNOR staining respectively, were significantly higher in LMS by comparison to UL, CL or AL. Because many singular cases had superimposed data being difficult to diagnose, a new scoring system for pathological evaluation was created. The results obtained by this scoring system suggest that immunohistochemical markers Ki-67 (MIB-1), bcl-2, p53 together with the AgNOR staining could be useful, by the scoring system, as an adjunct to the current accepted morphologic criteria in differentiating smooth muscle tumors of the uterus.     

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.     

苜蓿和扁蓿豆萌发期耐盐指标筛选及耐盐性综合评价

为探究苜蓿(Medicago sativa L.)和扁蓿豆(Medicago ruthenica(L.)Trautv.)种子萌发期的耐盐性强弱,采用复盐溶液模拟盐胁迫的方法,研究了不同浓度梯度(0.3%、0.6%、0.9%、1.2%、1.5%)盐胁迫对5份野生苜蓿和5份野生扁蓿豆种子萌发的影响。测定了发芽率、发芽势、发芽指数、活力指数、根长、苗长、幼苗干重,利用方差分析、相关分析、主成分分析、隶属函数分析和灰色关联分析进行了耐盐性综合评价。结果表明相同指标不同浓度盐溶液和不同材料之间多存在显著差异,各指标之间存在不同程度的相关关系。各指标中与耐盐强弱最相关的指标为幼苗干重,其关联度为0.752,其次为发芽率,关联度为0.745。供试10份材料中来源于中国内蒙古通辽市大青沟自然保护区的扁蓿豆和中国内蒙古呼和浩特武川县的扁蓿豆耐盐性较强,其综合耐盐D值和关联度均较高,分别为0.807、0.699和0.793、0.716。总体比较供试苜蓿和扁蓿豆材料,扁蓿豆萌发期耐盐性较强。     

Determination of optimal rehydration,fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.     

Leaf cell membrane stability‐based mechanisms of zinc nutrition in mitigating salinity stress in rice

• Excess salt affects about 955 million ha of arable land worldwide, and 49% of agricultural land is Zn‐deficient. Soil salinity and zinc deficiency can intensify plant abiotic stress. The mechanisms by which Zn can mitigate salinity effects on plant functions are not well understood.
• We conducted an experiment to determine how Zn and salinity effects on rice plant retention of Zn, K⁺ and the salt ion Na⁺ affect chlorophyll formation, leaf cell membrane stability and grain yield. We examined the mechanisms of Zn nutrition in mitigating salinity stress by examining plant physiology and nutrition. We used native Zn‐deficient soils (control), four salinity (EC ) and Zn treatments – Zn 10 mg·kg^?1 (Zn₁₀), EC 5 dS ·m^?1 (EC ₅), Zn₁₀+EC ₅ and Zn₁₅+EC ₅, a coarse rice (KS ‐282) and a fine rice (Basmati‐515) in the study.
• Our results showed that Zn alone (Zn₁₀) significantly increased rice tolerance to salinity stress by promoting Zn/K⁺ retention, inhibiting plant Na⁺ uptake and enhancing leaf cell membrane stability and chlorophyll formation in both rice cultivars in native alkaline, Zn‐deficient soils (P  <  0.05). Further, under the salinity treatment (EC ₅), Zn inputs (10–15 mg·kg^?1) could also significantly promote rice plant Zn/K⁺ retention and reduce plant Na⁺ uptake, and thus increased leaf cell membrane stability and grain yield. Coarse rice was more salinity‐tolerant than fine rice, having significantly higher Zn/K⁺ nutrient retention.
• The mechanistic basis of Zn nutrition in mitigating salinity impacts was through promoting plant Zn/K⁺ uptake and inhibiting plant Na⁺ uptake, which could result in increased plant physiological vigour, leaf cell membrane stability and rice productivity.

     

Photoactivation studies of zinc porphyrin-myoglobin system and its application for light-chemical energy conversion

An artificial zinc porphyrin-myoglobin-based photo-chemical energy conversion system, consisting of ZnPP-Mb or ZnPE(1)-Mb as a photosensitizer, NADP(+) as an electron acceptor, and triethanolamine as an electron donor, has been constructed to mimic photosystem I. The photoirradiated product is able to reduce a single-electron acceptor protein cytochrome c, but cannot catalyze the two-electron reduction of acetaldehyde by alcohol dehydrogenase, thus demonstrating a single electron transfer mechanism. Furthermore, the artificial system can bifunctionally promote oxidoredox reactions, depending on the presence or absence of a sacrificial electron donor, thus suggesting its potential application in electrochemical regeneration steps involved in chemical transformation and/or energy conversion.     

The glands of Tamarix aphylla: a system for salt recretion or for carbon concentration?

During periods of high atmospheric humidity, twigs of Tamarix aphylla (L.) Karst. become covered by an alkaline solution. The pH of that solution fluctuates between 8.0 – 8.5 in the dark and 10.5 during the light hours. Such a solution, produced by the glands, constitutes an efficient trap for atmospheric CO₂. Upon the periodic drop in pH, much of the preabsorbed carbon may gradually be released from the solution. This enriches the immediate surroundings of the twigs with CO₂ for prolonged periods of time. The expected concentrations of CO₂, at the boundary layer between the atmosphere and the surfaces of the twigs, are over 1 000 ppm. As net photosynthesis of T. aphylla reaches maximal rates only at CO₂ concentrations of above 500 ppm, the plants may benefit from this extra source of carbon and may exploit it for maximal assimilation during the early morning hours. Thus, the "salt glands'of Tamarix , which are liable for the production of the alkaline recretum, may serve a triple purpose: (a) removal of excess salts out of the twigs, (b) provision of a cover of hygroscopic solutes that moistens the twigs and shortens the duration of transpiration, and (c) providing the plants with an environment enriched in CO₂.     

Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H₂O₂ oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC₅₀ 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC₅₀ 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).     

Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface

PhoE protein is an abundant outer membrane protein of theEscherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foreign antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein ofMycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuatedSalmonella strains, which can be used as live oral vaccines, was shown.Paper awarded the Kluyver Prize 1990 by the Netherlands Society of Microbiology to Dr. M.C. Agterberg     

The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system

GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.     

Silver electrode as a sensor for determination of zinc in cell cultivation medium.

Use of the silver electrode as a sensor for the monitoring of zinc in cell growth medium is described. Zinc at silver electrodes provides specific voltammetric signal, which is affected by solution components. Signals of zinc ions in phosphate buffer solutions with and without cell growth medium were compared. Common DMEM cell culture medium was used for the cultivation of a cell line of v-myb-transformed chicken monoblasts and its variants expressing v-jun and c-jun in a zinc-dependent manner. Electrochemical results showed zinc concentrations in the medium coincide very well with the jun expression. With respect to the low toxicity of silver for eukaryotic cells, silver electrodes represent promising tools for the determination of zinc concentrations in vivo without the potential risk of a cell culture damage.     

Evaluation of chemical methods for estimating available zinc and response of green gram (Phaseolus aureus roxb) to applied zinc in non-calcareous soils

Summary Studies were conducted in 22 non-calcareous soils (India) to evaluate various extractants,viz. (6N HCl, 0.1N HCl, EDTA (NH₄)₂CO₃, EDTA NH₄OAc, DTPA+CaCl₂ and 1M MgCl₂) to find critical levels of soil and plant Zn for green gram (Phaseolus aureus Roxb.). The order of extractability by the different extractants was 6N HCl>0.1N HCl>EDTA (NH₄)₂CO₃<EDTA NH₄OAc DTPA+CaCl₂>1M MgCl₂. Critical levels of 0.48 ppm DTPA × CaCl₂ extractable Zn, 0.80 ppm EDTA NH₄OAc extractable Zn, 0.70 ppm EDTA (NH₄)₂CO₃ extractable Zn, and 2.2 ppm 0.1N HCl extractable Zn were estimated for the soils tested. The critical Zn concentration in 6 weeks old plants was found to be 19 ppm. The 0.1N HCl method gave the best correlation (r=0.588^**) between extractable Zn and Bray's per cent yield, while with DTPA+CaCl₂, it was slightly low (r=0.542^**). The DTPA + CaCl₂ method gave significant (r=0.73^**) correlation with plant Zn concentration. The 0.1N HCl gave the higher correlation with Zn uptake (r=0.661^**) than DTPA (r=0.634^**) 6N HCl and 1M MgCl₂ method gave nonsignificant positive relationship with Bray's per cent yield. For noncalcareous soils apart from the common use of DTPA+CaCl₂, 0.1N HCl can also be used for predicting soil available Zn. The use of 0.1N HCl would be much cheaper than DTPA and other extractants used in the study.     

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z′ factors were > 0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed,paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Dual‐wavelength excitation for fluorescence‐based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood

Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron‐restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual‐wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual‐wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (r_s = 0.943, p < 0.0001; range 37–689 μmol ZnPP/mol heme, 84–1238 nmol/L) and for PPIX (r_s = 0.959, p < 0.0001; range 42–4212 μmol PPIX/mol heme, 93–5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single‐wavelength excitation and with commercial front‐face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual‐wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)