Browsing by Subject "Immunofluorescence"
Now showing 1 - 9 of 9
Results Per Page
Sort Options
- PublicationOpen AccessButylated hydroxytoluene induces type-V collagen and overexpression of remodeling genes/proteins in experimental lung fibrosis(Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Martins, Vanessa; Teodoro, Walcy Rosolia; Pereira Velosa, Ana Paula; Andrade, Priscila; Farhat, Cecília; Fabro, Alexandre Todorovic; Capelozzi, Vera LuizaAnomalous histoarchitecture with increased levels of type-V collagen (Col V) in lungs of human idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM) airway-centered interstitial fibrosis suggest that this collagen can be a possible trigger involved in the pathogenesis of these diseases. Butylated hydroxytoluene (BHT) injury model revealed a distal involvement of lung parenchyma with significant endothelial injury and fibrotic response, contrasting with the BLM airway-centered insult. We undertook this study to analyze whether BHT alters distal airway/alveolar epithelial cells (AECs) and extracellular matrix (ECM) signaling involved in the initiation and progression of pulmonary fibrosis in a different pathway concerning overexpression of Col V. Female mice C57BL/6 (n=6) were instilled intraperitoneally with 400mg/kg of BHT dissolved in 1 mL of corn oil and euthanized at day 14 or 21 after BHT administration. Morphometry, immunohistochemistry and transmission electron microscopy were performed to characterize microscopic and submicroscopic changes of AECs and endothelial cells through transforming growth factor beta (TGF-β) basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression. Immunofluorescence and immunogold electron microscopy were performed to characterize Col V. Quantitative polymerase chain reaction (qPCR) was used to confirm differential levels of RNA messenger. BHT lungs showed marked fibrotic areas and hyperplastic AECs. The alveolar damage caused destruction of elastic fibers and a critical increase of Col V in ECM of distal lung parenchyma. Fibrogenesis-promoting markers TGF-β, bFGF and VEGF were also overexpressed in situ, coinciding with up-regulation in remodeling enzymes, growth factors, cytokines, transduction and transcription genes. BHT alters distal lung parenchyma signaling involved in pulmonary fibrosis highlighted similarities to human IPF in a pathway involving Col V arising as a promissory model to identify effective therapeutic targets.
- PublicationOpen AccessDouble immunofluorescence labeling for CD31 and CD105 as a marker for polyether polyurethane-induced angiogenesis in mice(Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Figueiredo, Camila Couto; Braga Pereira, Núbia; Pereira, Luciana Xavier; Machado Oliveira, Laser Antônio; Peixoto Campos, Paula; Passos Andrade, Silvia; Moro, LucianaA crucial component of the integration between foreign implants and the host is angiogenesis. However, to date, none of the available techniques and/or endothelial markers employed to assess angiogenesis in the implant/host interface seems to be able to highlight vascular structures convincingly. In the present study we investigated and compared the expression of two endothelial cell markers: platelet endothelial cell adhesion molecule (PECAM-1) (CD31) and endoglin (CD105) using immunohistochemistry (IHC) and immunofluorescence (IF) to identify and quantify newly formed blood vessels in subcutaneous implants of polyether-polyurethane sponge of formalin-fixed paraffin-embedded tissue. At day 14 post implantation the discs of the synthetic matrix were removed and processed for histological and morphometric analysis. In IHC staining for CD31 antibody the number of vessels was 2.27±0.69 and 5.25±0.46 for CD105. In IF for CD31 the number of vessels was 15.36±1.295 and 10.54±0.8213 for CD105. The level of cross-reaction was lesser in IF images compared with IHC images. Colocalization of CD31/CD105 using confocal images showed positive correlation (Pearson's co-relation and Manders’ equation). The double labeling for blood vessels using the IF technique for CD31/CD105 may be an important tool for evaluation of angiogenesis in biomaterial/host integration.
- PublicationOpen AccessDynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development(Murcia : F. Hernández, 2003) Unda, F.; Pérez-Nanclares, G.; Le Morvan, V.; Hernández, C.; Vilaxa, A.; De-la-Fuente, M.; Gorry, P.Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO- 1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.
- PublicationOpen AccessGap junctions and expression of Cx36 Cx43 and Cx45 in the posterodorsal medial amygdala of adult rats(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Zancan, Mariana; Malysz, Tais; Moura, Dinara J.; Morás, Ana Moira; Steffens, Luiza; Rasia-Filho, AlbertoThe posterodorsal medial amygdala (MePD) has a synaptic organization that dynamically modulates reproduction and other social behaviors in rats. Discrete gap junctions between glial cells were previously reported in the MePD neuropil. Connexins (Cx) are components of gap junctions and indicative of cellular electrical coupling. Here, we report the ultrastructural occurrence of gap junctions between neurons in the MePD and demonstrate the expression and immunofluorescent labeling of Cx36, Cx43 and Cx45 in this subcortical area of adult male rats. Few neuronal gap junctions were found in the MePD and, when identified, occurred between dendrites. On the other hand, there is a diffuse presence and distribution of punctate labelling for the tested Cxs. Puncta were visualized isolated or forming clusters in the same focal plane of cell bodies or along the MePD neuropil. The Cx36 puncta were found in neurons, Cx43 in astrocytes and Cx45 in both neurons and astrocytes. Our data indicate the presence of few gap junctions and different Cxs composition in the MePD. Because Cxs can assemble, form hemichannel units and/or serve as transcriptional regulator, it is likely that additional modulation of intercellular communication can occur besides the chemical transmission in the MePD of adult rats.
- PublicationOpen Accesslmmunofluorescent examination of the kidney post mortem(Murcia : F. Hernández, 1987) Lászik, Z.; Iványi, B.; Ormos, J.106 selected kidneys removed at autopsy were studied by direct immunofluorescence using polyvalent antisera against human immunoglobulins, light chains, complement fractions and fibrinogen. The immunofluorescence was a suitable me'thod to solve differential diagnostic problems that arose at autopsy. The diagnostic value was the most obvious in cases of immunologically mediated renal diseases and in immunologically mediated systemic diseases involving the kidneys. Negative immunofluorescence findings were also useful to determine the pathogenesis of renal lesions, especially in vasculopathies. The immunofluorescence of postmortem material showed similar disturbances to that obtained with biopsy material. At various sites, especially in the tubulo-interstitium, additional electron microscopical study was sometimes needed to localise the immune deposits exactly. The fluorescent microscopical examination of frozen sections of kidney taken at necropsy turned out to be more adequate than the immunoperoxidase examination of formalin-fixed, paraffin-embedded sections.
- PublicationOpen AccessLow temperature (15ºC) induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum in HeLa cells(Springer, 2007-08-11) Tomás, Mónica; Ballesta Germán, José; Martínez Alonso, Emma; Martínez Menárguez, José Ángel; Biología Celular e HistologíaLow temperature induces a transport blockade at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in cultured cells. Our previous studies support that the primary effect of low temperature is the detachment of COPI complexes from membranes. In the present study, we have used immunofluorescence and cryoimmunoelectron microscopy to investigate the effects of low temperature on both COPII and clathrin coat complexes in HeLa cells. Strikingly, COPII proteins moved from membranes to the cytosol at 15ºC, accumulating into electron-dense areas. In agreement with this observation, we also showed that ER exit is delayed in cells cultured at this temperature. By contrast, clathrin coat is not affected. Together, our results demonstrate that low temperature induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum.
- PublicationRestrictedMonitoring of Leishmania infantum in captive non-human primates in Spain(Elsevier, 2024-09-25) Barbero Moyano, Jesús; Martínez, Remigio; Beato Benítez, Adrián; Moreno, Inmaculada; Cano Terriza, David; Carretero, Andrea; Canales Merino, Rocío; Ferreiro Prado, Andrea; Risalde, María A.; Garrido, Juan J.; García Bocanegra, Ignacio; Gonzálvez Juan, Moisés; Sanidad Animal; Facultades de la UMU::Facultad de VeterinariaCases of Leishmania infantum infection have recently been reported in non-human primates (NHPs) in Spain causing severe clinical disease in critically endangered orangutans (Pongo pygmaeus pygmaeus). The aim of this study was to determine exposure and risk factors associated with L. infantum infection in NHPs housed in zoos and wildlife rescue centers (WRC) in Spain. Between 2007 and 2023, sera from 252 NHPs belonging to 47 different species were collected at 15 centers. Indirect immunofluorescence was used to detect the presence of antibodies against L. infantum (cut-off ≥1:80). In addition, hair samples from 78 individuals were tested for Leishmania kDNA by real-time quantitative PCR (qPCR). Anti-Leishmania antibodies were detected in 4.0 % (10/252; 95 %CI: 1.6–6.4) of the NHPs tested at 26.7 % (4/15) of the centers sampled. Twenty-two NHPs were longitudinally sampled between 2010 and 2023: one ring-tailed lemur (Lemur catta) seroconverted and a seropositive orangutan increased antibody titers during the study period. Leishmania infantum kDNA was found in 62.8 % (49/78; 95 %CI: 52.1–73.6) of animals and at all centers sampled (100 %; 7/7). Phylogenetic analysis revealed high homology between the sequence obtained and strains previously isolated in humans, dogs and captive and free-living wildlife species in Spain. To the authors´ knowledge, this is the first report of Leishmania kDNA detection in NHP hair samples. The results indicate that hair samples could be a useful, non-invasive method of detection of L. infantum infection in these species. This is also the first large-scale survey of L. infantum conducted in NHP species in Europe. We report for the first time the presence of Leishmania kDNA in nine different NHP species belonging to the families Cercopithecidae, Lemuridae, and Hylobatidae, expanding the host range for this parasite. The main risk factors associated with L. infantum infection were: age (≥5 years old) and body size (large). Our results demonstrate widespread circulation of this parasite among NHPs housed in Spain, which could be of conservation and public health concern. Monitoring and control programs should be implemented in zoos and WRCs to minimize the risk of NHP exposure to L. infantum in endemic areas worldwide.
- PublicationOpen AccessNeutrophil extracellular traps in tissue pathology(Universidad de Murcia. Departamento de Biología Celular e Histología, 2017) Nakazawa, Daigo; Kumar, Santhosh V.; Desai, Jyaysi; Anders, Hans JoachimNeutrophil extracellular traps (NETs) are innate immune systems against invading pathogens. NETs are characterized as released DNA mixed with cytoplasmic antimicrobial proteins such as myeloperoxidase, proteinase3 and neutrophil elastase. While NETs are thought to have an important role in host defense, recent work has suggested that NETs contribute to tissue injury in non-infectious disease states. Uncontrolled NET formation in autoimmune diseases, metabolic disorders, cancers and thrombotic diseases can exacerbate a disease or even be a major initiator of tissue injury. But spotting NETs in tissues is not easy. Here we review the available histopathological evidence on the presence of NETs in a variety of diseases. We discuss technical difficulties and potential sources of misinterpretation while trying to detect NETs in tissue samples
- PublicationOpen AccessSingle-cell spatial proteomics(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Liyanage, Senal; Guo, Jia; Biología Celular e HistologíaRecent advancements in single-cell spatial proteomics have revolutionized our ability to elucidate cellular signaling networks and their implications in health and disease. This review examines these cutting-edge technologies, focusing on mass spectrometry (MS) imaging and multiplexed immunofluorescence (mIF). Such approaches allow high-resolution protein profiling at the single-cell level, revealing intricate cellular heterogeneity, spatial organization, and protein functions within their native cellular contexts. MS imaging techniques offer unprecedented high-dimensional resolution and provide detailed insights into their subcellular protein localization and abundance. mIF enables rapid and high-throughput protein profiling, enhancing its accessibility for diverse research and clinical applications. This review assesses the current challenges associated with these methodologies and also discusses the potential solutions to overcome these obstacles. The integration of spatial proteomics with other systems biology approaches holds great promise for enhancing our understanding of complex biological systems. It could also lead to significant advancements in molecular diagnostics and personalized treatment strategies.