To analyse fluorescent fusion proteins, conidia of the respective strains were inoculated in 8-well ibidi-chambers or 60 μ-dishes (ibidi GmbH, Martinsried, Germany) containing AMM. Germ tubes were generated by overnight incubation at 30 °C. Fungal cells were then grown to the desired length and the samples were analysed using a Leica SP-5 microscope equipped with an environmental chamber adjusted to 37 °C (Leica Microsystems). To analyse fluorescent fusion proteins, conidia of the respective strains were inoculated in 8-well ibidi-chambers or 60 μ-dishes (ibidi GmbH) containing AMM. When the hyphae reached an appropriate length, they were analysed using a Leica SP-5 microscope equipped with an environmental chamber adjusted to 37 °C (Leica Microsystems, Wetzlar, Germay). All micrographs were taken using a Leica HCX PL APO lamda blue 63 × 1.4 Oil UV objective. Further image processing was performed using Adobe Photoshop CS. Time-lapse confocal images were recorded using a Leica SP-5 microscope under the conditions described above. The resulting image stacks were merged and exported as avi files using the SP-5 and ImageJ software. To quantify the time that a Woronin body remained at a certain septum, strains expressing GFP-HexA were analysed by live cell imaging. 30 septa per strain were analysed by generating image stacks every 2 min over a period of 1 h.
Leica sp5 microscope
Manufactured by Leica camera
Sourced in Germany
The Leica SP5 is a high-performance confocal laser scanning microscope designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research needs. The SP5 provides exceptional image quality, resolution, and sensitivity, enabling detailed analysis of biological and material samples.
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Lab products found in correlation
Leica sp5 confocal microscope, by Leica camera (3 mentions)
Ultracomp ebeads, by Thermo Fisher Scientific (2 mentions)
Leica sp8 sted microscope, by Leica camera (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Calcofluor, by Merck Group (1 mentions)
Ab37296, by Abcam (1 mentions)
L9393, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Sp 2002, by Vector Laboratories (1 mentions)
T8787, by Merck Group (1 mentions)
B 1325, by Vector Laboratories (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Jem 1200 exii electron microscope, by JEOL (1 mentions)
Anti lgr5, by Abcam (1 mentions)
Anti pan cytokeratin, by Merck Group (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Autoquant x3, by Media Cybernetics (1 mentions)
26 protocols using leica sp5 microscope
1
Fluorescent Fusion Protein Analysis
2
Cryptococcal Cell Labeling Protocol
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In some experiments, cryptococcal cells were labeled with mAb 18B7 labeled with Alexa-488 [78 (link)] at 1 μg/mL to label the capsule, and calcofluor (10 μg/mL, Sigma Aldrich) to label the cell wall. The cells were incubated for 1 h at 37, and then, they were washed and observed in a confocal SP5 Leica microscope.
3
Tau Protein Aggregation Assay
4
Immunofluorescence Staining of Kidney Organoids
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Kidneys organoid and LTL+ cells were washed with PBS. Next samples were fixed with 4% paraformaldehyde (153799, Aname) for 20 min at room temperature. Specimens were washed twice with PBS and further blocked using Tris-buffered saline (TBS) with 6% donkey serum (S30, Millipore) and 1% Triton X-100 (T8787, Sigma) for 1h at room temperature. After three rinses with antibody dilution buffer, samples were treated for 4h at room temperature with fluorescent conjugated secondary antibodies (Alexa Fluor (A) Cy3- or A647-; 1:200). A previous blocking step with a streptavidin/biotin blocking kit (SP-2002, Vector Labs) was performed for biotinylated LTL (B-1325, Vector Labs) and Alexa Fluor 488-conjugated streptavidin (SA5488, VectorLabs) to detect LTL+ cells. Antibodies to NEPHRIN (R&D SYSTEMS 4269; 1:100) and LAMININ (Sigma L9393; 1:50), SGLT2 (Abcam AB37296; 1:100), NaKATPase (Abcam; AB209299; 1:200) and SLC3A1 (Sigma HPA038360; 1:50) were used overnight at 4°C diluted in antibody dilution buffer consisting of TBS with 6% donkey serum and 0.5% Triton X-100. Nuclei were detected using 4,6-diamidino-2-phenylindole (DAPI; 1:5000, D1306, Life Technologies) for 30min. For mounting, samples were immersed in Fluoromount-G (0100-01, Southern Biotech). Sample confocal images were acquired with an SP5 Leica microscope and LTL + were analyzed using ImageJ.
5
Microscopic Analysis of Cultured Cells
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Cultured cells on collagen membranes were evaluated using an inverted light microscope (Nikon TI-Eclipse). For electron microscopy, cells were harvested by trypsinization at different time-points of culture, pelleted by spinning (6000xg for 10 min at 4°C), fixed and dehydrated according to standard procedures (Jurado et al. 1998) . Observations were performed using a JEM-1200 EX II Electron Microscope (Jeol, Tokyo, Japan).
Confocal microscopy was carried out in a SP5 Leica Microscope (Leica, Wetzlar, Germany). Cells were harvested from membranes by trypsinization at different DOC and citospinned on polylisine positive charged slides (Pearl, Carlsbad, CA, USA). Slides were fixed with acetone for 10 min, washed with PBS, and blocked with 2% BSA. Slides were incubated for 16 h with monoclonal antibodies anti-pan-cytokeratin (undiluted, Sigma-Aldrich, St. Louis, MO, USA), anti-LGR5
(1/100, Abcam, Cambridge, UK) or anti-vimentin (1/1000, Sigma-Aldrich, St. Louis, MO, USA) as primary antibody, followed with Alexa 488-conjugated anti-mouse γ globulin (1/2000) (Abcam, Cambridge, UK) for 1 h at C. Nuclei were counterstained with 1µg.ml -1 propidium iodide (PI).
Slides were mounted and visualized in the microscope.
Confocal microscopy was carried out in a SP5 Leica Microscope (Leica, Wetzlar, Germany). Cells were harvested from membranes by trypsinization at different DOC and citospinned on polylisine positive charged slides (Pearl, Carlsbad, CA, USA). Slides were fixed with acetone for 10 min, washed with PBS, and blocked with 2% BSA. Slides were incubated for 16 h with monoclonal antibodies anti-pan-cytokeratin (undiluted, Sigma-Aldrich, St. Louis, MO, USA), anti-LGR5
(1/100, Abcam, Cambridge, UK) or anti-vimentin (1/1000, Sigma-Aldrich, St. Louis, MO, USA) as primary antibody, followed with Alexa 488-conjugated anti-mouse γ globulin (1/2000) (Abcam, Cambridge, UK) for 1 h at C. Nuclei were counterstained with 1µg.ml -1 propidium iodide (PI).
Slides were mounted and visualized in the microscope.
6
Multicolor Confocal Imaging of Splenic Immune Cells
7
Quantitative Neuronal Morphometry
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Dendrites and cell body of fluorescent dentate neurons were traced using a computer-controlled microscope-based system (Axio Imager A2 Zeiss microscope, 100X oil-immersion objective) with a software (Neurolucida® software; MicroBrigthField Bioscience) that provides neuron tracing tools to trace from a live camera image (QImaging).
For spine analysis, confocal stacks of images were obtained with a Leica SP5 confocal microscope (63X oil-immersion objective; XY dimensions: 41.0 µm; z-axis interval: 0.13 µm). The dendritic length of each segment was measured on Z projections, and the number of dendritic spines was counted using NeuronStudio software [35 (link)]. Before spine analysis, images were deconvoluted using AutoQuantX3 software (Media Cybernetics). A minimum of 30 dendritic segments per experimental group and time point were examined for spine analysis.
Morphometric analysis of the AIS was done as previously described [36 ] using a Leica SP5 microscope (63X water-immersion objective; XY dimensions: 82.0 µm; z-axis interval: 0.21 µm).
For spine analysis, confocal stacks of images were obtained with a Leica SP5 confocal microscope (63X oil-immersion objective; XY dimensions: 41.0 µm; z-axis interval: 0.13 µm). The dendritic length of each segment was measured on Z projections, and the number of dendritic spines was counted using NeuronStudio software [35 (link)]. Before spine analysis, images were deconvoluted using AutoQuantX3 software (Media Cybernetics). A minimum of 30 dendritic segments per experimental group and time point were examined for spine analysis.
Morphometric analysis of the AIS was done as previously described [36 ] using a Leica SP5 microscope (63X water-immersion objective; XY dimensions: 82.0 µm; z-axis interval: 0.21 µm).
8
Multicolor Confocal Imaging of Immune Cells
9
Immunocytochemical analysis of neuronal markers
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For immunocytochemistry, cells were cultured on glass coverslips. After washing in PBS, cells were fixed 10 min at RT with PFA 4%. For Rnd2 staining, we used the PGT-based protocol described in “Immunohistochemistry section” with a fluorescent secondary antibody. For other stainings, cells were treated with PBS – 0.01% Triton X-100 – 1% bovine serum albumin (BSA, Sigma) for 30 min and incubated overnight at 4 °C with primary antibodies diluted in blocking solution: mouse anti-MAP2 (1/500, Sigma, M4403), chicken anti-nestin (1/400, Aves Labs, NES), mouse anti-Tuj1 (1/2000, Promega, G712A). Cells were then incubated with appropriate fluorescent secondary antibodies. Following this step, DAPI was added for 10 min. Images were acquired using an Eclipse Ti-U Nikon or a Leica SP5 microscope.
10
Immunofluorescence Assay for Protein Localization
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Cells were seeded in appropriate medium on cover slips in 12-well plates (BD Falcon, Le Pont-de-Claix, France). Once adherent, cells were fixed (formaldehyde, 4% in PBS, at RT, 15 min) permeabilized with PBS-0.05% saponin (20 min at RT) and saturated (BSA, 4% in PBS, 30 min). The cells were then successively incubated with primary antibodies to PRAAHG or to PAVIRF peptides, or with SAB L194 antibody, for 120 min at RT and secondary AlexaFluor488-labeled antibodies to rabbit IgG for 60 min. The cell nuclei were labeled with 1 μM Draq5 for 30 min. Confocal microscopy acquisitions were performed using a Leica SP5 microscope coupled with a Leica scanning device (Leica Microsystems, Mannheim, Germany). Images were recorded with LAS AF Lite acquisition software and were analysed with the public-domain ImageJ software (NIH; http://rsb.info.nih.gov/nih-image/ ).
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