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1.
J Anat ; 239(5): 1221-1225, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34633083

RESUMO

Teaching and learning anatomy by using human cadaveric specimens has been a foundation of medical and biomedical teaching for hundreds of years. Therefore, the majority of institutions that teach topographical anatomy rely on body donation programmes to provide specimens for both undergraduate and postgraduate teaching of gross anatomy. The COVID-19 pandemic has posed an unprecedented challenge to anatomy teaching because of the suspension of donor acceptance at most institutions. This was largely due to concerns about the potential transmissibility of the SARS-CoV-2 virus and the absence of data about the ability of embalming solutions to neutralise the virus. Twenty embalming solutions commonly used in institutions in the United Kingdom and Ireland were tested for their ability to neutralise SARS-CoV-2, using an established cytotoxicity assay. All embalming solutions tested neutralised SARS-CoV-2, with the majority of solutions being effective at high-working dilutions. These results suggest that successful embalming with the tested solutions can neutralise the SARS-CoV-2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching.


Assuntos
COVID-19/virologia , Transmissão de Doença Infecciosa/prevenção & controle , Embalsamamento/métodos , Formaldeído/farmacologia , Pandemias , SARS-CoV-2 , Fixação de Tecidos/métodos , COVID-19/transmissão , Cadáver , Células Cultivadas , Fixadores/farmacologia , Humanos
2.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502087

RESUMO

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.


Assuntos
Criopreservação/métodos , Sistema Hipotálamo-Hipofisário/citologia , Neurônios/ultraestrutura , Fixação de Tecidos/métodos , Animais , Criopreservação/normas , Feminino , Fixadores , Formaldeído , Macaca fuscata , Masculino , Microscopia Imunoeletrônica/métodos , Microscopia Imunoeletrônica/normas , Neurônios/metabolismo , Fixação de Tecidos/normas , Vasopressinas/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo
3.
Acta Cytol ; 65(6): 510-521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34535585

RESUMO

INTRODUCTION/OBJECTIVE: Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions. MATERIALS AND METHODS: Sediments of cultured RAJI cells (derived from Burkitt's lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used. RESULTS: For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted. CONCLUSIONS: Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted.


Assuntos
Antígenos CD20/análise , Antígenos/análise , Linfoma de Burkitt/imunologia , Fixadores/química , Imuno-Histoquímica , Antígeno Ki-67/análise , Antígenos Comuns de Leucócito/análise , Fixação de Tecidos , Especificidade de Anticorpos , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Humanos , Biópsia Líquida , Valor Preditivo dos Testes , Estabilidade Proteica , Fatores de Tempo
4.
J Immunol Methods ; 496: 113097, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34217694

RESUMO

Autofluorescence (AF) in formalin-fixed and paraffin-embedded tissues limit their use in immunofluorescence staining techniques. Various methods have been used to reduce AF in human and animal tissues but no protocol has been optimized for avian tissues. The present study was undertaken to evaluate different treatment methods including ammonium chloride, glycine, Trypan blue, sodium borohydride, Sudan Black B, potassium permanganate, LED light, cupric sulphate combined with glycine, ammonium chloride and cupric sulphate in reducing AF in FFPE chicken tissues for the detection of FITC labelled antibodies against immune cell markers. Chicken tissues including conjunctiva, trachea and Harderian gland presented intense non-homogenous AF in cells resembling erythrocytes, connective cells and melanocytes. Only Sudan Black B effectively reduced AF in FFPE tissues; however, no specific fluorescent signal was observed for six FITC labelled antibodies against immune cell markers. Specific fluorescent signal from the FITC-labelled antibodies was observed in frozen chicken tissue sections with minimal AF, suggesting that the AF in FFPE tissues is related to the use of formaldehyde fixatives. In conclusion, this study demonstrates for the first time that AF quenching methods commonly used for other animal species are not appropriate for use in avian tissues and that frozen tissue sections are recommended for immunofluorescence staining techniques in poultry.


Assuntos
Compostos Azo/química , Fixadores/química , Imunofluorescência , Formaldeído/química , Naftalenos/química , Fixação de Tecidos , Animais , Galinhas , Crioultramicrotomia , Fluoresceína-5-Isotiocianato/química , Fluorescência , Corantes Fluorescentes/química , Indicadores e Reagentes , Microscopia Confocal , Microscopia de Fluorescência , Inclusão em Parafina
5.
Anticancer Res ; 41(8): 3949-3953, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281858

RESUMO

BACKGROUND: To compare the number of lymph nodes (LNs) detected when using Carnoy's solution (CS) versus 10% neutral buffered formalin (NBF) to fix specimens after radical gastrectomy for gastric cancer. PATIENTS AND METHODS: LNs were routinely detected using NBF until 2020, since then, for the fixation procedure, residual fat was fixed in CS for 24 hours and dissected again for the detection of further LNs. Of 143 specimens, 117 were included in the NBF group and 26 in the CS group. RESULTS: The mean numbers of LNs examined were 27.85±14.89 and 36.30±12.41 in the NBF and CS groups, respectively (p=0.008). The mean number of additional LNs detected using CS was 8.07±2.91, of which 0.38±1.02 were metastatic. Additional LNs were found in all patients of the CS group, and all were ≤3 mm. Of the 26 patients in the CS group, metastatic LNs were detected in four, disease in two of whom was up-staged. CONCLUSION: CS is an appropriate alternative to NBF for the fixation of gastric cancer specimens, and more LNs were detected in the resected specimens fixed when using CS compared with NBF.


Assuntos
Ácido Acético , Clorofórmio , Etanol , Fixadores , Linfonodos/patologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Fixação de Tecidos/métodos , Idoso , Feminino , Formaldeído , Gastrectomia , Humanos , Masculino , Pessoa de Meia-Idade
6.
Biomolecules ; 11(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068806

RESUMO

Mitochondria are highly dynamic organelles, constantly undergoing shape changes, which are controlled by mitochondrial movement, fusion, and fission. Mitochondria play a pivotal role in various cellular processes under physiological and pathological conditions, including metabolism, superoxide generation, calcium homeostasis, and apoptosis. Abnormal mitochondrial morphology and mitochondrial protein expression are always closely related to the health status of cells. Analysis of mitochondrial morphology and mitochondrial protein expression in situ is widely used to reflect the abnormality of cell function in the chemical fixed sample. Paraformaldehyde (PFA), the most commonly used fixative in cellular immunostaining, still has disadvantages, including loss of antigenicity and disruption of morphology during fixation. We tested the effect of ethanol (ETHO), PFA, and glutaraldehyde (GA) fixation on cellular mitochondria. The results showed that 3% PFA and 1.5% GA (PFA-GA) combination reserved mitochondrial morphology better than them alone in situ in cells. Mitochondrial network and protein antigenicity were well maintained, indicated by preserved MitoTracker and mitochondrial immunostaining after PFA-GA fixation. Our results suggest that the PFA-GA combination is a valuable fixative for the study of mitochondria in situ.


Assuntos
Fixadores/farmacologia , Formaldeído/farmacologia , Glutaral/farmacologia , Mitocôndrias/ultraestrutura , Polímeros/farmacologia , Animais , Camundongos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos
7.
Sci Rep ; 11(1): 11640, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079006

RESUMO

Whole exome sequencing (WES) is used to identify mutations in a patient's tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/genética , Exoma , Frequência do Gene , Neoplasias Pulmonares/genética , Mutação , Alelos , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Fixadores , Formaldeído , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Técnicas de Amplificação de Ácido Nucleico , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Sequenciamento Completo do Exoma
8.
J Histochem Cytochem ; 69(6): 389-405, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34010071

RESUMO

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF-ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.


Assuntos
Fixação de Tecidos/métodos , DNA/análise , DNA/genética , Fixadores/química , Formaldeído/química , Humanos , Imuno-Histoquímica/métodos , Reação em Cadeia da Polimerase , RNA/análise , RNA/genética
9.
J Adv Res ; 30: 185-196, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34026295

RESUMO

Introduction: Chemotherapeutic drugs are the main intervention for cancer management, but many drawbacks impede their clinical applications. Nanoparticles as drug delivery systems (DDSs) offer much promise to solve these limitations. Objectives: A novel nanocarrier composed of red blood cell (RBC)-derived vesicles (RDVs) surface-linked with doxorubicin (Dox) using glutaraldehyde (glu) to form Dox-gluRDVs was investigated for improved cancer therapy. Methods: We investigated the in vivo antineoplastic performance of Dox-gluRDVs through intravenous (i.v.) administration in the mouse model bearing subcutaneous (s.c.) B16F10 tumor and examined the in vitro antitumor mechanism and efficacy in a panel of cancer cell lines. Results: Dox-gluRDVs can exert superior anticancer activity than free Dox in vitro and in vivo. Distinct from free Dox that is mainly located in the nucleus, but instead Dox-gluRDVs release and efficiently deliver the majority of their conjugated Dox into lysosomes. In vitro mechanism study reveals the critical role of lysosomal Dox accumulation-mediated mitochondrial ROS overproduction followed by the mitochondrial membrane potential loss and the activation of apoptotic signaling for superior anticancer activity of Dox-gluRDVs. Conclusion: This work demonstrates the great potential of RDVs to serve a biological DDS of Dox for systemic administration to improve conventional cancer chemotherapeutics.


Assuntos
Doxorrubicina/administração & dosagem , Eritrócitos/química , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Nanopartículas/química , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Feminino , Fixadores/química , Glutaral/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/uso terapêutico , Neoplasias/metabolismo , Espécies Reativas de Oxigênio
10.
Hum Reprod ; 36(7): 1871-1890, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33956944

RESUMO

STUDY QUESTION: Can ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin (NBF) with 5% acetic acid (referred to hereafter as Form-Acetic)? SUMMARY ANSWER: Fixation with Form-Acetic improved ovarian tissue histology compared to NBF in multiple species while still enabling histological molecular analyses. WHAT IS KNOWN ALREADY: NBF fixation results in tissue shrinkage in various tissue types including the ovary. Components of ovarian tissue, notably follicles, are particularly susceptible to NBF-induced morphological alterations and can lead to data misrepresentation. Bouin's solution (which contains 5% acetic acid) better preserves tissue architecture compared to NBF but is limited for immunohistochemical analyses. STUDY DESIGN, SIZE, DURATION: A comparison of routinely used fixatives, NBF and Bouin's, and a new fixative, Form-Acetic was carried out. Ovarian tissue was used from three different species: human (n = 5 patients), sheep (n = 3; 6 ovaries; 3 animals per condition) and mouse (n = 14 mice; 3 ovaries from 3 different animals per condition). PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue from humans (aged 13 weeks to 32 years), sheep (reproductively young i.e. 3-6 months) and mice (10 weeks old) were obtained and fixed in 2 ml NBF, Bouin's or Form-Acetic for 4, 8, and 24 h at room temperature. Tissues were embedded and sectioned. Five-micron sections were stained with haemotoxylin and eosin (H&E) and the percentage of artefact (clear space as a result of shrinkage) between ovarian structures was calculated. Additional histological staining using Periodic acid-Schiff and Masson's trichrome were performed on 8 and 24 h NBF, Bouin's and Form-Acetic fixed samples to assess the compatibility of the new fixative with stains. On ovarian tissue fixed for both 8 and 24 h in NBF and Form-Acetic, immunohistochemistry (IHC) studies to detect FOXO3a, FoxL2, collagen IV, laminin and anti-Müllerian hormone (AMH) proteins were performed in addition to the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay to determine the compatibility of Form-Acetic fixation with types of histological molecular analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Fixation in Form-Acetic improved ovarian tissue morphology compared to NBF from all three species and either slightly improved or was comparable to Bouin's for human, mouse and sheep tissues. Form-Acetic was compatible with H&E, Periodic acid-Schiff and Masson's trichrome staining and all proteins (FOXO3a, FoxL2, collagen IV and laminin and AMH) could be detected via IHC. Furthermore, Form-Acetic, unlike NBF, enabled antigen recognition for most of the proteins tested without the need for antigen retrieval. Form-Acetic also enabled the detection of damaged DNA via the TUNEL assay using fluorescence. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, IHC analysis was performed on a select number of protein types in ovarian tissue thus encouraging further studies to confirm the use of Form-Acetic in enabling the detection of a wider range of protein forms in addition to other tissue types. WIDER IMPLICATIONS OF THE FINDINGS: The simplicity in preparation of Form-Acetic and its superior preservative properties whilst enabling forms of histological molecular analyses make it a highly valuable tool for studying ovarian tissue. We, therefore, recommend that Form-Acetic replaces currently used fixatives and encourage others to introduce it into their research workflow. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Oxford Medical Research Council Doctoral Training Programme (Oxford MRC-DTP) grant awarded to B.D.B. (Grant no. MR/N013468/1), the Fondation Hoffmann supporting R.A. and the Petroleum Technology Development Fund (PTDF) awarded to B.V.A.


Assuntos
Formaldeído , Ovário , Preservação de Tecido/métodos , Ácido Acético , Animais , Hormônio Antimülleriano , Feminino , Fixadores , Humanos , Imuno-Histoquímica , Camundongos , Ovinos
11.
Methods Mol Biol ; 2271: 303-316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908016

RESUMO

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.


Assuntos
Fixadores/química , Formaldeído/química , Glicoproteínas/análise , Glicosídeo Hidrolases/metabolismo , Inclusão em Parafina , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fixação de Tecidos , Animais , Configuração de Carboidratos , Glicosilação , Humanos , Isomerismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Projetos de Pesquisa , Especificidade por Substrato , Fluxo de Trabalho
12.
RNA ; 27(6): 725-733, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33846273

RESUMO

The mammalian cell nucleus contains different types of membrane-less nuclear bodies (NBs) consisting of proteins and RNAs. Microscopic imaging has been widely applied to study the organization and structure of NBs. However, current fixation methods are not optimized for such imaging: When a fixation method is chosen to maximize the quality of the RNA fluorescence in situ hybridization (FISH), it often limits the labeling efficiency of proteins or affects the ultrastructure of NBs. Here, we report that addition of glyoxal (GO) into the classical paraformaldehyde (PFA) fixation step not only improves FISH signals for RNAs in NBs via augmented permeability of the fixed nucleus and enhanced accessibility of probes, but also largely preserves protein fluorescent signals during fixation and immunostaining. We also show that GO/PFA fixation enables the covisualization of different types of nuclear bodies with minimal impact on their ultrastructures under super-resolution microscopy.


Assuntos
Estruturas do Núcleo Celular/ultraestrutura , Fixadores/química , Formaldeído/química , Glioxal/química , Hibridização in Situ Fluorescente/métodos , Polímeros/química , Células HEK293 , Células HeLa , Humanos
13.
Methods Appl Fluoresc ; 9(3)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33853048

RESUMO

The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2needed for 100% fixation is 50µg ml-1, which is much lower than that of traditional fixatives (1000-10000µg ml-1). The ClO2mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO2fixation on the staining. The results demonstrated that ClO2fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO2has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.


Assuntos
Proteínas de Bactérias/química , Compostos Clorados/química , Reagentes para Ligações Cruzadas/química , Fixadores/química , Óxidos/química , Escherichia coli O157/química , Imunofluorescência , Química Verde , Coloração e Rotulagem
14.
Sci Rep ; 11(1): 7595, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828141

RESUMO

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.


Assuntos
Inclusão em Parafina/métodos , Manejo de Espécimes/métodos , Fixação de Tecidos/métodos , Biomarcadores Tumorais/genética , Cromatografia Líquida/métodos , Fixadores/química , Formaldeído/química , Humanos , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Preservação de Tecido/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/genética
15.
Methods Mol Biol ; 2259: 25-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687707

RESUMO

Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions-such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.-are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enable the protein profiling of these small and diverse subcellular structures.


Assuntos
Extensões da Superfície Celular/química , Microdissecção e Captura a Laser/métodos , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Animais , Linhagem Celular , Fixadores , Humanos
16.
Anticancer Res ; 41(3): 1341-1348, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33788725

RESUMO

BACKGROUND/AIM: Cancer profiling tests using formalin-fixed paraffin-embedded (FFPE) specimens with various conditions have become an essential tool for cancer treatment. The robustness of these tests needs to be addressed. MATERIALS AND METHODS: A cancer profiling test, NCC oncopanel, was tested with FFPE specimens from various tissues with different storage conditions and fixation lengths. Next generation sequencing was performed with Miseq and the data were assembled using the human reference genome hg19. RESULTS: Duration of storage and fixation affected the mapping statistics. Prolonged storage increased outward read paring and longer fixation rates caused increased singletons and unmapped reads. CONCLUSION: Our results indicate that a cancer profiling test with target capturing method, NCC oncopanel, shows robustness for FFPE cancer specimens with various storage conditions.


Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Inclusão em Parafina/métodos , Manejo de Espécimes/métodos , Fixação de Tecidos/métodos , Fixadores/química , Formaldeído/química , Genômica/métodos , Humanos , Mutação , Neoplasias/patologia
17.
Forensic Sci Int Genet ; 52: 102485, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33636658

RESUMO

Immunolabeling is a technique, which has recently been introduced to enhance the quality of developed fingermarks and subsequently strengthen the evidential value. The effect of this method on subsequent DNA analysis, however, has not been explored yet. Therefore, the current pilot study aimed to determine whether STR profiling is possible after immunolabeling. Since immunolabeling involves washing steps which could reduce DNA quantities, the use of different fixatives including methanol, formaldehyde and universal molecular fixative (UMFIX) were investigated. STR profiles from the (immunolabeled) fingermarks were generated after four days and four weeks by a direct PCR method to enable comparison of relatively fresh and old fingermarks. The fingermarks were deposited on diverse forensically relevant substrates, including glass, metal and tile. STR profiles could be recovered for all tested fixatives with no significant difference in performance. However, the mean number of detected alleles was the highest when methanol was used for fixation. Furthermore, immunolabeling on aged fingermarks (4 weeks) was also possible, but the number of detected alleles showed a non-significant decrease. DNA could be recovered from deposits on all substrates, of which glass showed the highest mean number of detected alleles followed by metal and tile.


Assuntos
Impressões Digitais de DNA , Dermatoglifia , Imuno-Histoquímica , Repetições de Microssatélites , Feminino , Fixadores , Formaldeído , Humanos , Masculino , Metanol , Projetos Piloto , Reação em Cadeia da Polimerase
18.
Sci Rep ; 11(1): 3150, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542368

RESUMO

Segmentation of axons in light and electron micrographs allows for quantitative high-resolution analysis of nervous tissues, but varied axonal dispersion angles result in over-estimates of fiber sizes. To overcome this technical challenge, we developed a novel shape-adjusted ellipse (SAE) determination of axonal size and myelination as an all-inclusive and non-biased tool to correct for oblique nerve fiber presentations. Our new resource was validated by light and electron microscopy against traditional methods of determining nerve fiber size and myelination in rhesus macaques as a model system. We performed detailed segmental mapping and characterized the morphological signatures of autonomic and motor fibers in primate lumbosacral ventral roots (VRs). An en bloc inter-subject variability for the preganglionic parasympathetic fibers within the L7-S2 VRs was determined. The SAE approach allows for morphological ground truth data collection and assignment of individual axons to functional phenotypes with direct implications for fiber mapping and neuromodulation studies.


Assuntos
Axônios/ultraestrutura , Microscopia Eletrônica/normas , Fibras Nervosas Mielinizadas/ultraestrutura , Raízes Nervosas Espinhais/ultraestrutura , Animais , Axônios/fisiologia , Feminino , Fixadores , Formaldeído , Glutaral , Região Lombossacral/inervação , Macaca mulatta , Microscopia Eletrônica/métodos , Fibras Nervosas Mielinizadas/fisiologia , Polímeros , Raízes Nervosas Espinhais/fisiologia , Fixação de Tecidos/métodos
19.
Pharmacol Res Perspect ; 9(1): e00716, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33523576

RESUMO

Glutaraldehyde-fixed porcine heart valve (GPHV) calcify and deteriorate over time. The aim of this study was to explore the roles macrophages play in mediating calcification and degeneration of the valve's connective tissue matrix. GPHV were implanted subcutaneously in the abdomens of C57BL/6 mice. The mice were equally divided into two study groups: (a) GPHV +phosphate buffered saline (PBS) liposomes, and (b) GPHV +clodronate liposomes. GPHV were collected for further analyses at 4 weeks post implant. Macrophages were almost depleted from the spleens of mice injected with clodronate liposomes as indicated by immunohistochemical staining. Furthermore, the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and proinflammatory cytokines like IL-1ß, IL-6, MCP-1, MIP-1a, MIP-1b, were downregulated in the GPHV +Clodronate liposomal group compared with the GPHV+PBS liposomal group. Clodronate liposomal treatment led to significant decreases in the expression of RUNX2, ALP and OPN as well as less calcium deposits in GPHVs compared with PBS liposomal treatment. This finding indicated that infiltrating macrophages are critically involved in the development of calcification and deterioration in GPHVs. Macrophage depletion by clodronate liposomes decreased the extent of GPHV's calcification and deterioration.


Assuntos
Bioprótese , Ácido Clodrônico/administração & dosagem , Próteses Valvulares Cardíacas , Macrófagos Peritoneais/efeitos dos fármacos , Falha de Prótese/efeitos dos fármacos , Animais , Valva Aórtica/metabolismo , Calcinose/prevenção & controle , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Fixadores , Glutaral , Lipossomos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Suínos , Linfócitos T/efeitos dos fármacos
20.
J Vis Exp ; (168)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33616116

RESUMO

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.


Assuntos
Vasos Sanguíneos/fisiologia , Pulmão/fisiologia , Perfusão , Pressão , Alvéolos Pulmonares/fisiologia , Animais , Fixadores , Camundongos
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