Super‐resolution confocal image was performed with an inverted Leica SP8X WLL microscope, equipped with a pulsed WLL2 laser (470–670 nm) and acusto‐optical beam splitter. 0.8 μm thick image stacks at a z‐step size of 200 nm were acquired with a 100×/1.4 NA oil immersion objective, scan speed was 100 Hz, and image pixel size was set to 39 nm. The pinhole was set to 0.6 AU (calculated for 633 nm). The following fluorescence settings were used: B23, Alexa Fluor 488: (excitation: 488 nm; emission: 496–535 nm), Ncl, Alexa Fluor 546: (561; 569–600), and Trnp1, Abberior STAR 635P: (633; 643–701). Recording was done line sequentially to avoid bleed‐through. A triple notch filter (NF 488/561/633) was used to block reflected excitation light. All signals were recorded with hybrid photodetectors (HyDs) in counting mode. Stacks were deconvolved using Huygens Professional 17.10 p2. Images shown are single planes of deconvolved stacks.
Sp8x wll microscope
Manufactured by Leica
The SP8X WLL microscope is a versatile imaging system designed for advanced fluorescence microscopy. It features a white light laser (WLL) that provides a wide range of excitation wavelengths, enabling researchers to image a variety of fluorescent samples. The SP8X WLL microscope delivers high-quality, high-resolution images to support scientific investigations.
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Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
Prolong diamond antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
A7811, by Merck Group (1 mentions)
Anti α actinin, by Merck Group (1 mentions)
Anti mouse alexa546, by Thermo Fisher Scientific (1 mentions)
C6219, by Merck Group (1 mentions)
A 11030, by Thermo Fisher Scientific (1 mentions)
Vectamount aq, by Vector Laboratories (1 mentions)
A21441, by Thermo Fisher Scientific (1 mentions)
Anti n cadherin 610921, by BD (1 mentions)
A5228, by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Micro well, by MatTek (1 mentions)
Evolve 512, by Zeiss (1 mentions)
Alpha plan apo 100 1.46 oil dic m27 objective, by Zeiss (1 mentions)
4 protocols using sp8x wll microscope
1
Super-resolution Confocal Imaging Protocol
2
Phagocytosis of E. coli by Neutrophils
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Heparinized human whole blood pre-treated with PAP-1 (10 nM) or vehicle and murine whole blood from WT and KV1.3−/− mice was incubated with Escherichia coli bio particles (pHrodo Green E. coli BioParticle Phagocytosis Kit, Thermo Fisher Scientific) for 30 min at 37°C. Phagocytosis was stopped and cells were fixed according to the manufacturer’s protocol. As control, whole blood was incubated for 30 min at 4°C. Samples were analysed using a Beckman Coulter Gallios flow cytometer and Kaluza Flow analysis Software (Beckman Coulter). Ly6G+ and CD15+/CD66b+ positive populations were defined as neutrophils, respectively.
For confocal microscopy, phagocytosis was stopped by putting E. coli particles-treated whole blood on ice. Neutrophils were isolated (on ice) using EasySep neutrophil enrichment kit. Cells were then seeded on poly-L-lysine (0.1%; Sigma-Aldrich) coated object slides (Ibidi), fixed with 2% PFA washed and stained with DAPI (Invitrogen) for 5 min at RT before embedding them in ProLong Diamond Antifade mounting medium (Invitrogen). Samples were imaged by confocal microscopy at the core facility Bioimaging of the Biomedical Center with a Leica SP8X WLL microscope, equipped with a HC PL APO 40×/1.30 NA oil immersion objective. Images were processed (including removal of outliers and background subtraction) using FIJI software.
For confocal microscopy, phagocytosis was stopped by putting E. coli particles-treated whole blood on ice. Neutrophils were isolated (on ice) using EasySep neutrophil enrichment kit. Cells were then seeded on poly-L-lysine (0.1%; Sigma-Aldrich) coated object slides (Ibidi), fixed with 2% PFA washed and stained with DAPI (Invitrogen) for 5 min at RT before embedding them in ProLong Diamond Antifade mounting medium (Invitrogen). Samples were imaged by confocal microscopy at the core facility Bioimaging of the Biomedical Center with a Leica SP8X WLL microscope, equipped with a HC PL APO 40×/1.30 NA oil immersion objective. Images were processed (including removal of outliers and background subtraction) using FIJI software.
3
Detailed Immunohistochemistry of Heart Tissue Slices
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Thin paraffin sections of tissue slices were stained with hematoxylin–eosin using standard techniques. Immunohistochemistry was done on whole-mount heart slices that had been fixed in 4% paraformaldehyde for at least 24 h. The tissues were equilibrated with a graded series of 4, 15 and 30% sucrose in PBS, and were permeabilized with 1% Triton X-100 overnight. After blocking (3% BSA in PBS, 12 h), samples were incubated sequentially with primary antibodies (anti-α-actinin, A7811, anti-connexin-43, C6219, anti-α-smooth muscle actin, A5228, all Sigma-Aldrich, anti-vimentin, AB92547, Abcam, anti-N-cadherin, #610921, BD-Biosciences, all 1:100), and secondary antibodies (anti-rabbit-Alexa488, A21441, anti-mouse-Alexa546, A11030, both ThermoFisher, 1:100, combined with DNA-stain 1 µM TO-PRO-3) for 1 day each. Washing steps between all incubations used citrate-buffer (150 mM NaCl, 15 mM Na3-citrate, pH 7.2) supplemented with 1% BSA, 0.05% Triton X-100 and 3 mM NaN3. Slices were mounted in VectaMount AQ (Vector Laboratories). Confocal microscopy was performed at the bioimaging core facility of the Biomedical Center using an inverted Leica SP8X WLL microscope.
4
Live-cell Imaging of Cellular Foci
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Live-cell imaging was essentially performed as described38 (link). In brief, cells were grown overnight on rich medium (YES) to the logarithmic phase. Prior to imaging, cells were attached with lectin (Sigma) to glass-bottom dishes with a micro well (MatTek). Cells were imaged on a Zeiss AxioObserver Z1 confocal spinning disk microscope with an EMM-CCD camera (Photometrics, Evolve 512) through a Zeiss Alpha Plan/Apo ×100/1.46 oil DIC M27 objective lens. Z-stacks were obtained at focus intervals of 0.4 μm. FiJi/ImageJ software was used to measure the distances between the foci and the periphery.
For the imaging of cells expressing CFP-Mmi1, the following setup was used: confocal microscopy was performed at the Core Facility Bioimaging of the Biomedical Center (LMU Munich) with an inverted Leica SP8 X WLL microscope, equipped with 405-nm laser, WLL2 laser (470–670 nm), and acusto-optical beam splitter. Images were acquired with a HC PL APO ×93/1.30 GLYC motCORR-STED WHITE objective, and Z-stacks were obtained at focus intervals of 0.25 μm. Images were deconvolved using the SVI Huygens suite and FiJi/ ImageJ software was used to measure the distances between the foci and the periphery.
For the imaging of cells expressing CFP-Mmi1, the following setup was used: confocal microscopy was performed at the Core Facility Bioimaging of the Biomedical Center (LMU Munich) with an inverted Leica SP8 X WLL microscope, equipped with 405-nm laser, WLL2 laser (470–670 nm), and acusto-optical beam splitter. Images were acquired with a HC PL APO ×93/1.30 GLYC motCORR-STED WHITE objective, and Z-stacks were obtained at focus intervals of 0.25 μm. Images were deconvolved using the SVI Huygens suite and FiJi/ ImageJ software was used to measure the distances between the foci and the periphery.
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