Confocal microscopy was performed on 10-μm sections of renal frozen tissues or on cells by using a Leica SP5 AOBS confocal microscope (Leica) equipped with a Chameleon Ultra-II two-photon laser (Coherent). Detailed procedures are reported in Supplemental Experimental Procedures .
Sp5 aobs confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica SP5 AOBS confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features an Acousto-Optical Beam Splitter (AOBS) for flexible and efficient light management, enabling precise control over the excitation and detection of fluorescent samples. The SP5 AOBS provides researchers with a powerful tool for obtaining high-quality, detailed images of their specimens.
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Lab products found in correlation
Lasaf 2, by Leica (2 mentions)
Las af lite, by Leica (2 mentions)
Fluoromount g mounting medium, by Southern Biotech (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Recombinant cxcl16, by Thermo Fisher Scientific (2 mentions)
Pkh26, by Merck Group (2 mentions)
Pkh67, by Merck Group (2 mentions)
8 well chamber, by Ibidi (1 mentions)
Hcx pl apo cs, by Leica (1 mentions)
Axio scan z1, by Olympus (1 mentions)
Spinning disk confocal microscope, by Zeiss (1 mentions)
Las x software, by Leica (1 mentions)
Sglt2, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Illustrator cc, by Adobe (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rompun, by Bayer (1 mentions)
Las af, by Leica (1 mentions)
31 protocols using sp5 aobs confocal microscope
1
Confocal Microscopy of Renal Tissues
2
Live Cell Imaging of Ferroptosis
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Live cell imaging of iGPX4KD cells was performed as described before25 (link). Briefly, cells (untreated or stimulated for ferroptosis by removal of Fer1) have been seeded 10 × 103 cells/well in 8 well chamber (#80826, iBidi) in 250 µl of medium in the presence of DRAQ7 (3 µM) and the images were acquired on Leica Sp5 AOBS confocal microscope (Leica), using an ×40 HCX PL Apo UV 1.25 na oil objective. Obtained images were further analyzed and extracted using Fiji 1.53 built on ImageJ 2.1.0 software.
3
Live-cell Imaging of Apoptosis
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B16 cells were seeded at 15 × 103 cells per well in an 8-well chamber (iBidi) in 200 µl of complete growth medium. Twenty four hours later, cells were transfected with saline or 1 µg of mRNA encoding Fluc, tBid, or MLKL just before imaging. Live cell imaging was performed on a Leica Sp5 AOBS confocal microscope (Leica), using an ×40 HCX PL Apo UV 1.25 na oil objective. Images were acquired in a sequential mode every 30 min.
4
Quantifying Microglial Dynamics with Fluorescence Microscopy
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Fluorescence microscopy was performed using a confocal spinning disk microscope (Zeiss, Germany). Double-positive cells (Ki67+ and Iba-1+ positive) were counted on 6–12 representative images (267.20 × 267.73 µm) for each sample and an average was calculated. Bright-field microscopy was done using an olympus light microscope (BX51) and with a Axio Scan.Z1 (Zeiss, Germany). For 3D reconstruction of microglia, 70-μm vibratome sections from adult brain tissue were stained overnight with anti-Iba-1 (1:500) at 4 °C, followed by Dylight 594-conjugated secondary antibody (1:1000) overnight at 4 °C. Nuclei were counterstained with Hoechst. Confocal images were taken with a Leica Sp5 AOBS confocal microscope (Leica, Mannhein, Germany), using a HCX PL APO CS 40.0 × 1.25 UV oil objective. Three cells per mouse and five mice per condition were reconstructed. For live cell imaging, primary cultured microglia were seeded in an eight-well chamber (iBidi). Cell death was monitored by propidium iodide (PI) or Sytox Green uptake. Live cell imaging was performed on a Zeiss confocal spinning disk (Zeiss, Germany). Analysis was performed on Fiji, a public domain imaging software.
5
Fluorescent Labeling of Renal Cells
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Both hRPCs and hRPTECs were fluorescently labelled with antibodies directed against markers for differentiated epithelia and transport proteins. Briefly, cell-loaded membranes were fixed with 10% neutral buffered formalin for 1h at 4°C and then washed in PBS. The blocking step was performed for 30 minutes incubating the membranes in 3% bovine serum albumin (BSA, Sigma Aldrich) solution. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Epithelial protein analysis was evaluated with antibodies against the sodium-dependent glucose transporter (SGLT2, abcam, 1:30), aquaporin-1 (AQP-1, abcam, 1:100), chloride channel Ka (CLC-NKA, Santa Cruz Biotechnology, 1:25) and Na/Cl co-transporter (NCC, Santa Cruz Biotechnology, 1:25). Samples were washed in PBS and incubated with fluorescent labelled secondary antibodies Alexa Fluor 488 against the specific primary antibody species at 1:500 concentration. DAPI (Sigma Aldrich, 1:1000) was incubated along with the secondary antibody. After several rinses in PBS, constructs were mounted with VECTASHIELD (Vector Laboratories) and analysed under Leica SP5 AOBS confocal microscope (Leica, Wetzlar, Germany) equipped with a Chameleon Ultra-II two-photon laser (Coherent, Milan, Italy). Images were recorded digitally and further processed using LASX software (Leica).
6
Multicolor Confocal Imaging Protocol
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Images were acquired using a Leica SP5 AOBS confocal microscope (Leica Microsystems) equipped with a 40× (1.25 NA) oil immersion objective. Four hundred five–nanometer and five hundred sixty-one–nanometer lasers were used to excite DAPI and DsRed, respectively. Sequential acquisition of the multicolor images was used to avoid cross-excitation, and images were overlaid with the Leica LAS AF 2.6 software.
7
Multicolor Confocal Imaging Protocol
8
Immunofluorescence Imaging of Transfected HEK293 Cells
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HEK293 cells were seeded onto coverslips (18 mm) coated with poly-L-lysine, transfected as described above and fixed in 3% paraformaldehyde for 30 min. Immunostaining was performed as described previously (17 ). Results were evaluated with an SP5 AOBS confocal microscope (Leica Microsystems) with 63×/1.4 NA NAHCX Plan-Apochromat oil immersion objective, ∼1.2 airy unit pinhole aperture, and appropriate filter combinations. Images were acquired with 405 diode and argon ion/argon krypton lasers (Leica) and processed using LAS AF Lite (Leica), Photoshop CC, and Adobe Illustrator CC (Adobe Systems).
9
Confocal Microscopy of Frozen Sections
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Confocal microscopy was performed on 10-μm frozen sections by using an SP5 AOBS confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with a Chameleon Ultra II 2-photon laser (Coherent, Santa Clara, CA). Detailed procedures are given in Supplementary Procedures .
10
Immunofluorescence Analysis of Mouse Organs
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Deeply anesthetized mice were perfused via cardiac puncture with 4% paraformaldehyde in PBS. Harvested organs were incubated in 30% sucrose in PBS for at least 24 h before being embedded in Tissue-Tek OCT compound (#4583,Sakura Finetek, Alphen aan den Rijn, The Netherlands) and cut as 7μm-thick sections. Sections were incubated 1 h with blocking buffer (NP-40 0.5%, BSA 2%, normal goat serum (NGS) 3% in PBS) at room temperature, followed by primary antibody incubation overnight at 4°C. After washing three times with PBS, sections were incubated 1 h at room temperature with secondary antibodies and then washed four times with PBS. The sections were then mounted using Fluoromount-G mounting medium (#0100–01, Southern Biotech, Birmingham, USA). Fluorescent images were visualized using a laser scanning confocal microscope (SP5 AOBS Confocal Microscope, Leica Microsystems, Wetzlar, Germany).
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