COMPARISON OF TWO STAINING METHODS FOR ANODIZING IN ALLOY 6063 ALUMINUM PROFILES

Aluminum stands out for being a light, corrosion-resistant, and recyclable metal, achieving wide coverage in the market. When incorporated into alloying elements, it is possible to acquire other desirable characteristics. Alloy 6063, intended for architectural purposes, has aesthetic, structural, and strength functions. This study aims to compare two different staining methods on the surface of anodized profiles of aluminum alloy 6063. Anodized finishing is performed through an electrolytic process using sulfuric acid as an electrolyte to change the surface layer of the material, ensuring a more resistant aluminum oxide film than that formed naturally. For decorative purposes, the anodic film coloration can be performed by several methodologies, including, in this case, the coloration by organic adsorption, with the use of aniline, and the electrolytic coloration, composed of tin sulfate salts, both for obtaining the black color. To compare, neutral saline mist tests, scanning electron microscopy analysis, determination of the anodic layer thickness, and immersion tests with 3.5 percent sodium chloride for 1000 hours. The results obtained highlight that both were shown to be resistant to corrosion due to the fact that they do not present corrosion points when exposed to the neutral saline mist test for 600 hours. In the immersion tests, both remained resistant to sodium chloride. Because both methodologies present satisfactory results in all tests, the quality of the applied stains is ensured, and it is found that they are equivalent when the parameters discussed are used.

Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Immunocytochemical Labelling of Haematological Samples Using Monoclonal Antibodies

I reflect on my experience working with David Y. Mason in the Leukaemia Research Laboratories in the Nuffield Department of Pathology at the University of Oxford in the early 1980s. This was soon after the first monoclonal antibodies had been produced, which led to an exciting and productive time in biological discovery and pathology diagnostics. A specific focus in the laboratory was the development of immunoenzymatic staining methods that would enable monoclonal antibodies to be applied in diagnostic practice. This paper describes the work that led to the performance of immuno-alkaline phosphatase staining on blood and bone marrow smears, the success of which changed leukaemia diagnosis.

Techniques for visualizing fibroblast-vessel interactions in the developing and adult CNS

Fibroblasts are found associated with blood vessels in various locations across the CNS: in the meninges, the choroid plexus, and in the parenchyma within perivascular spaces. CNS fibroblasts have been characterized using transcriptional profiling and a Col1a1-GFP mouse line used to identify CNS fibroblasts in vivo. However, current methods for visualizing CNS fibroblasts are lacking and, in particular, prevent adequate assessment of fibroblast-vessel interactions. Here, we describe methods for whole mount visualization of meningeal and choroid plexus fibroblasts, and optical tissue clearing methods for visualization of parenchymal vessel-associated fibroblasts. Importantly, these techniques can be combined with immunohistochemistry methods for labeling different cell types in the meninges and blood vasculature as well as EdU-based cell proliferation assays. These methods are ideal for visualization of vessel-fibroblast interactions in these CNS structures and provide significant improvement over traditional sectioning and staining methods. We expect these methods will advance studies of CNS fibroblast development and functions in homeostasis, injury, and disease.

Histochemistry for nanomedicine: Novelty in tradition

During the last two centuries, histochemistry has provided significant advancements in many fields of life sciences. After a period of neglect due to the great development of biomolecular techniques, the histochemical approach has been reappraised and is now widely applied in the field of nanomedicine. In fact, the novel nanoconstructs intended for biomedical purposes must be visualized to test their interaction with tissue and cell components. To this aim, several long-established staining methods have been re-discovered and re-interpreted in an unconventional way for unequivocal identification of nanoparticulates at both light and transmission electron microscopy.

Effects of thymulin 5cH in granuloma evolution and B1 cell differentiation: an experimental model to understand its biological mechanisms

In previous studies, we found that thymulin (a thymic hormone), when prepared in homeopathic 5cH potency, had the property to improve the productive performance of broiler chickens infected with reovirus, as well as modulate the development of Ehrlich tumor and granuloma inflammatory lesions in mice by immune-mediated mechanisms. The aim of the present work was to study the immunomodulatory mechanisms of thymulin 5cH in a granuloma experimental model, by subcutaneous inoculation of BCG in mice, focusing the B-1 cells and zinc involvement in this process. Three groups of male Balb/c SPF mice (group A treated with thymulin 5cH, group B treated with thymulin 5cH incubated in Chelex ® - a zinc chelant - and group C, control, treated with vehicle) were inoculated with BCG in the left footpad and subcutaneous granuloma and spleen were harvested for histomorphometry analysis, after 7, 14 and 21 days. Ziehl-Neelsen, HE and Prussia Blue staining methods were used. Flow cytometry was also used in the same times to characterize and quantify peritoneal cells. Positive cells for CD11b (activated phagocytes, B-1 cells), CD19 (B-1 and B-2 cells), CD23 (negative B-1 cells, positive B2 cells) and CD5 (B-1a cells) were analyzed in a FACS Calibur (BD) device. Statistical analysis was performed using Kruskal - Wallis / Dunn for nonparametric evaluations and ANOVA / Tuckey-Krammer for the parametric ones. The X² method was used to evaluate the cell count in flow cytometry. P values ≤ 0.05 were considered statistically significant. Mice treated with thymulin 5cH presented higher macrophage activity and increase in the follicular area were seen in spleen after 7 days. Increase in gross lesion diameter and decrease in local BCG infection were seen after 21 days. At this time, the flow cytometry demonstrated the increase in peritoneal phagocytes derived from B-1 cells in thymulin 5cH treated mice, independently of Chelex ® incubation. The incubation of thymulin 5cH with Chelex ® blocked its effects only upon the number of B2 cells in the peritoneum and reduces Mn levels in the medicine solution. We conclude that thymulin 5CH modulates the BCG-induced granuloma through more than one mechanism, especially by peritoneal B1 cell differentiation into phagocytes.

Potential of Flow Cytometric Approaches for Rapid Microbial Detection and Chracterization in the Food Industry—A Review

As microbial contamination is persistent within the food and bioindustries and foodborne infections are still a significant cause of death, the detection, monitoring, and characterization of pathogens and spoilage microorganisms are of great importance. However, the current methods do not meet all relevant criteria. They either show (i) inadequate sensitivity, rapidity, and effectiveness; (ii) a high workload and time requirement; or (iii) difficulties in differentiating between viable and non-viable cells. Flow cytometry (FCM) represents an approach to overcome such limitations. Thus, this comprehensive literature review focuses on the potential of FCM and fluorescence in situ hybridization (FISH) for food and bioindustry applications. First, the principles of FCM and FISH and basic staining methods are discussed, and critical areas for microbial contamination, including abiotic and biotic surfaces, water, and air, are characterized. State-of-the-art non-specific FCM and specific FISH approaches are described, and their limitations are highlighted. One such limitation is the use of toxic and mutagenic fluorochromes and probes. Alternative staining and hybridization approaches are presented, along with other strategies to overcome the current challenges. Further research needs are outlined in order to make FCM and FISH even more suitable monitoring and detection tools for food quality and safety and environmental and clinical approaches.

COMPARISON OF TWO STAINING METHODS FOR ANODIZING IN ALLOY 6063 ALUMINUM PROFILES

Background: Aluminum stands out for being a light, corrosion-resistant, and recyclable metal, achieving wide coverage in the market. When incorporated into alloying elements, it is possible to acquire other desirable characteristics. Alloy 6063, intended for architectural purposes, has aesthetic, structural, and strength functions. Anodized finishing is performed through an electrolytic process, ensuring a more resistant aluminum oxide film than that formed naturally. For decorative purposes, the anodic film coloration can be performed by several methodologies, in this case, for the coloration by organic adsorption, with the use of aniline, and the electrolytic coloration, composed of tin sulfate salts, both for obtaining the black color. Aim: Compare of two different staining methods on the surface of anodized profiles of aluminum alloy 6063. Methods: Profile samples were collected and tests were carried out to measure the thickness of the anodic layer, immersion tests with 3,5 percent sodium chloride, for 1000 hours, and neutral saline mist, for 600 hours. Results and Discussion: Both methodologies proved to be resistant to immersion tests with sodium chloride, as well as with neutral saline mist, and these tests are quite aggressive and provide corrosion of the material when not well treated. Corrosion points were only seen at the intersections performed, and in the rest of thearea, no points were detected. Conclusions: The result of both methodologies was positive, considering tht there was no corrosion in the tested samples, except in the intersections performed, as well as the maintenance of the color in both tested methodologies, which was not expected in the literature. For future work, it is suggested to deepen the study to perform electrochemical impedance spectroscopy tests for exaluate the strength of the anodic film and perform anodizing with the same parameters, however, with different anilines to analyze their behavior.

Cajanol Sensitizes A2780/Taxol Cells to Paclitaxel by Inhibiting the PI3K/Akt/NF-κB Signaling Pathway

Ovarian cancer is the second most common gynecological malignancy, and one of the most deadly. The bottleneck restricting the treatment of ovarian cancer is its multi-drug resistance to chemotherapy. Cajanol is an isoflavone from pigeon pea (Cajanus cajan) that has been reported to have anti-tumor activity. In this work, we evaluate the effect of cajanol in reversing paclitaxel resistance of the A2780/Taxol ovarian cancer cell line in vitro and in vivo, and we discuss its mechanism of action. We found that 8 μM cajanol significantly restored the sensitivity of A2780/Taxol cells to paclitaxel, and in vivo experiments demonstrated that the combination of 0.5 mM/kg paclitaxel and 2 mM/kg cajanol significantly inhibited the growth of A2780/Taxol metastatic tumors in mice. Flow cytometry, fluorescence quantitative PCR, western blotting and immunohistochemical staining methods were used to study the mechanism of reversing paclitaxel resistance with cajanol. First, we determined that cajanol inhibits paclitaxel efflux in A2780/Taxol cells by down-regulating permeability glycoprotein (P-gp) expression, and further found that cajanol can inhibit P-gp transcription and translation through the PI3K/Akt/NF-κB pathway. The results of this work are expected to provide a new candidate compound for the development of paclitaxel sensitizers.

Ferruginol Restores SIRT1-PGC-1α-Mediated Mitochondrial Biogenesis and Fatty Acid Oxidation for the Treatment of DOX-Induced Cardiotoxicity

Background: Doxorubicin (DOX), a broad-spectrum chemotherapy drug, has life-threatening cardiotoxicity. Therefore, searching cardioprotective drugs for DOX-induced cardiotoxicity (DIC) is urgently needed.Objectives: This study aimed to explore cardioprotective effect and specific mechanism by which Ferruginol (FGL) attenuated DIC in vivo and in vitro.Methods: We evaluated the cardioprotection of FGL and performed high-throughput RNA-Seq on a DIC mouse. Whereafter, multiple methods, including western blot, RT-qPCR, a transmission electron microscope, CO-IP, immunofluorescence, and other staining methods, and antagonist of SIRT1 and PGC-1α were utilized to confirm the cardioprotection and molecular mechanism of FGL.Results: FGL-exerted cardioprotection manifested as enhanced cardiac function and reduced structural damage and apoptosis. The transcriptome and other results revealed that FGL facilitated PGC-1α-mediated mitochondrial biogenesis and fatty acid oxidation (MB and FAO) by increasing the expression of PGC-1α and concurrently promoting the expression of SIRT1-enhancing deacetylase SIRT1 deacetylating and activating PGC-1α.Conclusions: These results documented that FGL exerted cardioprotective effects restoring MB&FAO via the SIRT1–PGC-1α axis.