To prepare samples for confocal microscopy analysis, 4 cm2 blocks of R5 solid cultures were cut out using a scalpel and sliced using a microtome (sections with a width of approx. 0.15 mm). To characterize isolated hyphae, mycelium was scrapped from R5 solid cultures and resuspended in NaCl 0,9% (w/v). 10 μl of the mycelium resuspension were used for staining. Viability staining was performed using the “LIVE/DEAD BacLight Bacterial Viability Kit” (Molecular Probes) with a mixture of SYTO9 and propidium iodide in a 1:1 proportion and following the manufacturer instructions. Samples were observed under a Leica SP2 AOBS SE confocal laser-scanning microscope using 488 nm and 568 nm as excitation wavelengths and emitting between 520–545 nm (green) or 620–640 nm (red), as previously described6 (link). Micrographs were treated using the software ImageJ.
Sp2 aobs se
Manufactured by Leica
Sourced in Germany, United States
The SP2 AOBS SE is a confocal laser scanning microscope made by Leica. It is designed for high-resolution, multi-color imaging of fluorescent samples. The system features an acousto-optical beam splitter (AOBS) that allows for flexible wavelength selection and control. The SP2 AOBS SE provides researchers with a versatile tool for advanced microscopy applications.
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Lab products found in correlation
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Anti map2, by Abcam (1 mentions)
Anti tau, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 protocols using sp2 aobs se
1
Confocal Microscopy Analysis of Fungal Cultures
2
Immunofluorescence Staining of Neurons
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Cells were fixed in 4% sucrose/paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. Neurons were then incubated with 5% bovine serum albumin (Sigma) in PBS+0.1% Tween 20, for 1 h at 37 °C, to block nonspecific binding, and incubated with primary antibodies, overnight at 4 °C. Cells were then washed five times with PBS+ 0.1%Tween+ 0.5% bovine serum albumin and incubated with the appropriate secondary antibodies, for 1 h at 37 °C. The coverslips were mounted in a fluorescent mounting medium (DAKO) and imaging was performed on a laser scanning Confocal Microscope Leica (Wetzlar, Germany) SP2 AOBS SE, using the × 40/ × 63 oil objective. Primary antibodies used were anti-GFP (1:250, Santa Cruz, Dallas, TX, USA), anti-megalin (1:200, Biorbyt (Cambridge, UK), orb6173), anti-MAP2 (1:800; Abcam, ab24640), anti-Tau (1:750, Cell Signaling, #4019), as secondary antibodies Alexa Fluor 488 and 594 (1:750, Life technologies) were used. The fluorescent dye Hoechst 33342 (0.5 μg/ml–10 min room temperature) was used to stain nuclei.
3
Phagocytosis Assay of C. albicans
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Phagocytosis was assessed by flow cytometry following a previously described method [40] . Briefly, fixed C. albicans cells were labeled with Sytox Green and incubated in Dulbecco's modified Eagle medium (DMEM) during 30 min at 37 °C with 20% mouse serum collected from mice immunized thrice with CWSP, ADS1, ADS2 or EL. Serum from four independent mice was used, each one in triplicate. Then, cells were washed twice with PBS and resuspended in DMEM without serum. RAW 264.7 macrophages were incubated with labeled yeast suspensions at a multiplicity of infection (MOI) of 1 macrophage per 5 yeast cells, for 30 min, at 37 °C and 5% CO 2 . After incubation, plates were kept on ice to stop phagocytosis, and wells rinsed twice with PBS to remove unbound yeasts. Macrophages and associated yeasts were then incubated with PI at a final concentration of 6 lg/ml, for 5 min at RT. The percentage of macrophages with internalized cells (Sytox + PI À and Sytox + PI + ), as well as the percentage of macrophages with adherent cells (Sytox À PI + ), was determined as previously described [40] . Phagocytosis was confirmed by confocal microscopy (Leica SP2 AOBS SE) and images were analyzed using Fiji-ImageJ software 2.00 (NIH-USA).
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