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Identification of FXPRLamide Family Neuropeptides from the Japanese Oak Silkworm,Antheraea yamamai Using immunocytochemistry Methods

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Agricultural Sciences in China 2006年第12期5卷 944-951页

作者： WEI Zhao-jun LAI Juan-hong ZHAO Yuan Department of Biotechnology Hefei University of Technology Hefei 230009 P.R.China The Sericultural Research Institute Chinese Academy of Agricultural Sciences Zhenjiang 212018 P.R.China

In the present study, zooblooting, ELISA, and whole-mount immunocytochemistry methods were used to identify the FXPRLamide family neuropeptides from the Japanese oak silkworm, Antheraea yamamai. The results showed that the genomic DNA from A. yamamai showed positive bands after being hybridized with the fragment of DH-PBAN cDNA from Samia cynthia ricini, which was labeled with [α-32p]-dCTP. The SG showed highest FXPRLamide peptides titer in neural organs. Using an antiserum against Helicoverpa armigera PBAN, PBAN-like immunoreactivity was detected in the SG and TG of A. yamamai by whole-mount immunocytochemistry, and there were three cluster cells in the SG which shows positive PBAN-like immunoreactivity. The titers of FXPRLamide peptides immunoreactivity in the hemolymph were kept at a steady level. During pupation, the titer was increased promptly, but then decreased to a low level after the early pupal stage. The above-mentioned results demonstrate the existence of FXPRLamide family peptides in A. yamamai, but its function needs to be further investigated in the future.

关键词： Antheraea yamamai FXPRLamide family neuropeptides immunocytochemistry identification

ORIGINAL ARTICLES APPLICATION OF SILVER ACETATE AUTOMETALLOGRAPHY AND GOLD-SILVER STAINING METHODS FOR IN SITU DNA HYBRIDIZATION

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中华医学杂志(英文版) 1993年第2期106卷 83-92页

作者： GW Hacker AH Graf C Hauser-Kronberger G Wirnsberger A Schiechl G Bernatzky U Wittauer 苏慧慈 H Adam J Thurner G Danscher L Grimelius Landeskrankenanstalten Salzburg Institute of Pathological AnatomyImmunohistochemistry and Biochemistry UnitSalzburgAustria University of Graz Institute of PatholOgical AnatomyGraz.Austria University of Salzburg Institute of ZoologySalzburgAuatria University of Salzburg Institute of ZoologySalzburgAustria Department of Histology and Embryology Fourth Military Medical UniversityXi'anShaanxiPR China University of Salzburg Institute of ZoologySalzburgAuStria Landeskrankenanstalten Salzburg Institute Of Pathological AnatomyyImmunohistochemistry and Biochemistry UnitSalzburgAustria University of Aarhus Institute of AnatomyDepartment of NeurobiologyAarhusDenmark Uppsala University Hospital Institute of PathologyUppsalaSweden

In situ hybridization using biotinylated DNA probes hasbecome an important tool in *** is well knownthat the sensitivity of the methods used to demonstrate viralDNA in formalin-fixed and paraffin-embedded specimen de-pends strongly on the detection system *** the presentstudy,an optimized in situ DNA hybridization protocol wascombined with four different approaches of gold-silver *** silver enhancement,the recently described meth-od of silver acetate autometallography,a technique allowinghighly efficient development without the necessity of dark roomillumination has been *** most efficient detection methodfound in our experiments was the use of gold-adsorbedanti-biotin antibodies with subsequent silver *** procedure can be completed in 5 hours includinghybridization and is a highly sensitive alternative to peroxidaseand alkaline phosphatase detection systems.

关键词： MESSENGER-RNA ELECTRON-MICROSCOPY PARAFFIN SECTIONS DETECTION SYSTEMS GENE-EXPRESSION CDNA PROBE VIRUS-DNA IMMUNOGOLD TISSUE immunocytochemistry

Aberrant cytological localization of p16 and CDK4 in colorectal epithelia in the normal adenoma carcinoma sequence

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World Journal of Gastroenterology 2006年第39期12卷 6391-6396页

作者： PO Zhao Xin Mao Ian C Talbot Department of Pathology Chinese PLA General Hospital Beijing 100853 China Academic Department of Pathology St. Mark's Hospital Cancer Research UK Colorectal Cancer Unit Harrow HA1 3UJ London United Kingdom

AIM： To study the correlation between the patterns of subcellular expression of p16 and CDK4 in colorectal epithelia in the normal-adenoma-carcinoma sequence.METHODS： Paraffin sections of 43 cases of normal colorectal epithelia and corresponding adenomas as well as carcinomas were analysed immunocytochemically for subcellular expression of p16 and CDK4 proteins.RESULTS： Most carcinomas showed more cytoplasmic overexpression for p16 and CDK4 than the adenomas from which they arised or the adjacent normal mucosa. Most normal or non-neoplastic epithelia showed more p16 and CDK4 expression in the nucleus than their adjacent adenomas and carcinomas. There was a significant difference between the subcellular expression pattern of p16 and CDK4 in normal-adenoma-carcinoma sequence epithelia （P 〈 0.001）. Neither p16 nor CDK4 subcellular patterns correlated with histological grade or Dukes＇ stage.CONCLUSION： Interaction of expression of p16 and CDK4 plays an important role in the Rb/p16 *** of p16 and CDK4 in the cytoplasm, as well as loss expression of p16 in the nucleusmighlc be important in the evolution of colorectal carcinoma from adenoma and, of adenoma from normal epitheiia.

关键词： Colorectal neoplasm p16 CDK4 immunocytochemistry

Preparation of monoclonal antibody against human KIAA0100 protein and Northern blot analysis of human KIAA0100 gene

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Journal of Pharmaceutical Analysis 2017年第3期7卷 190-195页

作者： He Cui Xi Lan Shemin Lu Fujun Zhang Wanggang Zhang Department of Clinical Hematology The Affiliated Hospital Inner Mongolia Medical University Department of Biochemistry and Molecular Biology School of Basic Medical Sciences Xi'an Jiaotong University Health Science Center Department of Clinical Hematology The Second Affiliated No.2 Hospital Xi'an Jiaotong University

Monoclonal antibodies(MAbs) are important tools for the study of proteins′ function and structure. But there has been no report on the preparation of MAbs against human KIAA0100 protein up to date. Here, first, we generated the mouse MAb against human KIAA0100 protein using purified recombinant 6×Histidinc(6×His)-tagged human KIAA0100 protein segment(1557–2234) as an antigen; then, the m RNA expression of human KIAA0100 gene was detected in U937 cells using Northern blot analysis. The results showed that the mouse MAb against human KIAA0100 protein could sensitively recognize the human KIAA0100 protein using Western blot analysis and immunocytochemistry analysis. Besides, Western blot analysis revealed that human KIAA0100 gene possibly encoded two different protein products(254 k Da and < 250 k Da) in U937 cells. Moreover,Northern blot analysis confirmed that human KIAA0100 gene might produced two different m RNA products(6000–10000 bp and 5000–6000 bp) in U937 cells. The results provide a basis for large-scale production of the MAb against human KIAA0100 protein, which will be useful for the study of human KIAA0100 protein′s function/structure and MAb-targeted drugs in the future.

关键词： KIAA0100 Monoclonal antibody Northern blot Western blot immunocytochemistry

Three-Dimensional Sub-100 nm Super-Resolution Imaging of Biological Samples Using a Phase Ramp in the Objective Pupil

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Nano Research 2011年第6期4卷 589-598页

作者： David Baddeley Mark B. Cannell Christian Soeller Department of Physiology University of Auckland Private Bag 92019 Auckland 1142 New Zealand

Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.

关键词： Single molecules nanoscopy point-spread function engineering immunocytochemistry localisation microscopy

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

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Neural Regeneration Research 2014年第9期9卷 978-985页

作者： Zhiqiang Li Qingming Shu Lingzhi Li Maolin Ge Yongliang Zhang Second Department of Medicine Inner Mongolia Corps Hospital Chinese People’s Armed Police Forces Department of Pathology General Hospital of Chinese People’s Armed Police Forces Section of Pharmaceutical Chemistry Department of Rescue Medicine Logistics University of Chinese People’s Armed Police Force Second Department of Surgery Inner Mongolia Corps Hospital Chinese People’s Armed Police Forces Training Department Logistics University of Chinese People’s Armed Police Force Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott’s method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury.

关键词： nerve regeneration brain injury platelet activating factor cyclooxygenase-2 RT-PCR immunocytochemistry hippocampus platelet activating factor receptor glutamate receptor 2 NSFC grant neural regeneration

Biological properties of neonatal mouse olfactory ensheathing cells purified by differential attachment,cytosine arabinoside,and mitogen stimulation

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Neural Regeneration Research 2009年第8期4卷 629-634页

作者： Nan Zhao Jun Liu Hong Gao Gang Ma Jiangxia Pang Zhongtang Feng Tinghua Wang Department of Neurosurgery Kunming First People's Hospital Kunming 650011 Yunnan Province China Institute of Neuroscience Kunming Medical College Kunming 650031 Yunnan Province China

BACKGROUND： Differential attachment, chemicals, and immunoaffinity absorption are frequently used to purify olfactory ensheathing cells （OECs）. Although purity is high （〉 90%）, the complex process, high cost, decreased cell activity, and cell loss limit their application. OBJECTIVE： To purify OECs using differential attachment, cytosine arabinoside （Ara-C）, and mitogen stimulation, and to analyze the biological characteristics of OECs. DESIGN, TIME AND SETTING： Molecular biology experiment of cell morphology and immunocytochemistry. The study was performed at the Institute of Neuroscience, Kunming Medical College between January 2005 and January 2007. MATERIALS： N2 was purchased from Gibico, USA; basic fibroblast growth factor （bFGF） from Invitrogen, USA; PCR master mix kit from Fermentas, USA; nerve growth factor （NGF） and brain-derived neurotrophic factor （BDNF） from Santa Cruz Biotechnology, USA; OEC specific immunological marker NGF receptor （p75NGFR） from ABCAM, UK; immunological markers of oligodendrocyte and Schwann cells, cyclic nucleotide 3＇ phosphohydrolase （CNPase）, from NeoMarkers, USA; inverted fluorescence microscope from Leica, Germany. METHODS： OECs were isolated from olfactory bulbs of mice provided by Institute of Cancer Research （ICR mice） at postnatal 1 2 days, and purified by differential attachment, Ara-C inhibition （5 mg/L）, and 10 μg/L mitogen bFGF and 0.5% N2 stimulation. MAIN OUTCOME MEASURES： OEC growth was observed under inverted microscope; cell purity, as well as expression of NGF and BDNF, was determined by means of immunocytochemistry; expression of β-NGF, BDNF, NT-3, platelet-derived growth factor-B （PDGF-B）, bFGF, epidermal growth factor （EGF）, NGF receptor TrkA, BDNF receptor TrkB, and NT-4 mRNA were detected by RT-PCR. RESULTS： The majority of in vitro cultured OECs was bipolar or tripolar, and purity was estimated to be 〉 92.4%. immunocytochemistry demonstrated expression of p75NGFR, NGF, BDNF and CNPase. The RT-PCR results sugge

关键词： olfactory ensheathing cells cell culture immunocytochemistry RT-PCR

Immunocytochemical identification and localization of APUD cells in the gut of seven stomachless teleost fishes

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World Journal of Gastroenterology 2000年第1期6卷 96-101页

作者： Pan QS Fang ZP Zhao YX College of Fisheries Huazhong Agricultural UniversityWuhan 430070Hubei ProvinceChina College of Animal Husbandry and Veterinary Medicine Huazhong Agricultural UniversityWuhan 430070Hubei ProvinceChina

AIM To study the cell types,localization,distribution density and morphology of APUDcells in the intestinal mucosa of stomachlessteleost *** By using the peroxidase-antiperoxidase complex(PAP)immunocytochemical staining technique theidentification,localization and morphology ofimmunoreactive(IR)endocrine cells seattered inthe intestinal mucosa of grass carp(Cyenopharyngodon idellus),black carp(Mylopharyngodon piceus)and common carp(Cyprinus carpio)were investigated with 20kinds of antisera prepared against mammalianpeptide hormones of APUD cells,and likewise byusing avidin-biotin-peroxidase complex(ABC)method those of silver carp(Hypophthalmichthys molitrix),bighead(Aristichthys nobilis),silver crucian carp(Carassius gibelio)and bluntnose black bream(Megalobrama amblyocephala)were alsostudied with 5 different *** of the first antiserum by phosphatebuffered saline(PBS)was employed as a *** endocrine cells were counted with asquare-mesh ocular micrometer from 10 fieldsselected randomly in every section of each partof the intestine *** average numberof IR endocrine cells per mm2 was counted toquantify their distribution *** Gastrin(GAS)-,Gastric inhibitorypeptide(GIP)-,glucagon(GLU)-,glucagon-likeimmunoreactants(GLI)-,bovine pancreaticpolypeptide(BPP)-,leucine-enkephalin(ENK)-and substance P(SP)-IR endocrine cells werefound in the gut of grass carp,black carp andcommon carp,and somatostatin(SOM)-IRendocrine cells were only seen in common ***-,GIP-and GLU-IR endocrine cells werefound in the intestinal mucosa of silver carp,bighead,silver crucian carp and bluntnose *** of IR endocrine cells had the higherdistribution density in the foregut and midgut,and were longer in *** had a long apicalcytoplasmic process extended to the gut lumenand a basal process extended to adjacent cellsor basement membrane and touched with ***,the basal cytoplasmic processformed an enlarged synapse-like st

关键词： Subject headings stomachless teleost fishes APUD cells intestinal mucosa immunocytochemistry

KAI1 is a potential target for anti-metastasis in pancreatic cancer cells

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World Journal of Gastroenterology 2008年第7期14卷 1126-1132页

作者： Jian-Hua Xu Xiao-Zhong Guo Li-Nan Ren Li-Chun Shao Min-Pei Liu Department of Gastroenterology The General Hospital of Shenyang Military Command Shenyang 110016 Liaoning Province China

AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer ***: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t ***: By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively (P < 0.05).CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of

关键词： KAI1 Pancreatic cancer cell line Transfection immunocytochemistry Western blotting Immunofluorescence Gelatinolysis