eclipse ti e c2 confocal laser scanning microscope  (Nikon)

Journal: Biofilm

Article Title: Inactivation of Cysteine Synthase CysK-A enhances flocculation, biofilm formation, and sensitivity to oxidative stress in Azospirillum brasilense Sp7

doi: 10.1016/j.bioflm.2025.100335

Figure Lengend Snippet: Colony and biofilm calcofluor staining assays. Production of exopolysaccharide (EPS) by the wild-type ( A. brasilense Sp7), the cysK-A mutant ( A. brasilense AR), and the complemented ( A . brasilense AR-pAB- cysK ) strains were visualized by staining with calcofluor, which binds to β-linked polysaccharides. ( A ) Colonies were grown for 5 days at 30 °C on solid LB medium (lacking NaCl) supplemented with Calcofluor (200 μg/mL). Images were captured under UV illumination to visualize fluorescence. Scale bar = 10 mm. ( B ) Confocal micrographs of biofilms grown for 5 days at 30 °C on agar-solidified Nfb∗ medium containing Calcofluor (85 μM). The biofilms were observed with a Nikon Eclipse Ti-E C2+ microscope. Scale bar = 20 μm.

Article Snippet: After five days of static incubation at 30 °C, three-dimensional biofilm structures were observed using an Eclipse Ti-E C2+ confocal laser scanning microscope (Nikon) equipped with CFI Plan Apo VC 20x/1.2 and CFI Plan Apo VC 60x/1.2 WI objective lenses.

Techniques: Staining, Mutagenesis, Fluorescence, Microscopy

Journal: Communications Biology

Article Title: A role of pigment epithelium-derived factor in zinc-mediated mechanism of neurodegeneration in glaucoma

doi: 10.1038/s42003-025-08370-8

Figure Lengend Snippet: Effect of zinc stress on PEDF secretion was studied in ARPE−19 ( a ) and Y79 ( b ) cells. a APRE−19 shows a slow response to zinc stress (half-maximum after 24 h), as confirmed by MTT test, as well as fluorescein diacetate/propidium iodide (FDA/PI) viability assay and confocal microscopy (Scale bar represents 25 μm, n = 3). b Zinc treatment decreased viability (half-maximal after 8 h) and induced apoptosis of Y79 cells, as evidenced by EZ4U test and flow cytometric annexin A5 assay ( n = 6). In both cases, zinc stress was accompanied by PEDF release ( p < 0.05). The amount of PEDF in the absence (Y79) or at 24 h (ARPE−19) of stress was taken as 100%. c-e Effect of zinc on the neurotrophic activity of PEDF was studied on SH-SY5Y cells ( c ) and Y79 c ells ( d-e ). c Preincubation of PEDF with zinc reduced its axogenic activity in respect to differentiated SH-SY5Y cells without affecting cell count (Scale bar represents 25 μm, n = 14). d Zinc inhibited the ability of PEDF to stimulate neuronal differentiation of Y79 cells (Scale bar represents 10 μm, n = 21). Neurites and nuclei were stained with anti-β-III tubulin ( c ) or synapsin ( d ) antibodies and Hoechst 33342, respectively, and visualized by confocal microscopy. e PEDF att e nuated Y79 cell death under oxidative stress but not zinc stress, as shown by LDH assay ( n = 3). Leaked LDH activity in the absence of stress was taken as 100%. f Phospholipase activity of PEDF-R in ARPE−19 cell membranes was monitored in the presence of apo or zinc-bound PEDF by quantifying fatty acid release from model phospholipid substrate using HPLC-MS/MS. Extracted ion chromatogram illustrating differences in linoleic acid ([M-H] - LA, m/z 279.2) formation in the presence of apo and zinc-bound PEDF. The histogram on the right illustrates the attenuation of PEDF-mediated enhancement of PEDF-R activity in the presence of zinc ( p < 0.05). PEDF-R activity without PEDF/zinc was taken as 100%. All data are presented as mean and error bars indicate SEM.

Article Snippet: The staining solution, containing 5 ml DMEM, 8 μl FDA (5 mg/ml) and 50 μl PI (2 mg/ml) was added to the wells, cells were incubated at room temperature for 4-5 minutes in the dark, washed with PBS and examined on Eclipse Ti-E confocal laser scanning microscope (CLSM) with A1 confocal module (Nikon Corporation, Japan) and CFI Plan Apo VC 20×/0.75 objective lens with a life support system, maintaining normal culturing conditions (37°C, 5% CO 2 ).

Techniques: Viability Assay, Confocal Microscopy, Activity Assay, Cell Counting, Staining, Lactate Dehydrogenase Assay, Tandem Mass Spectroscopy