High-resolution images of METs structure were obtained by Zeiss Axio Observer equipped with LSM880 confocal microscopy system. The z-stack images of 4 neighboring microscopic fields were obtained using Plan-Apochromat 40×/0.95 Korr M27 objective lens and stitched to make a big image that can visualize the whole structure of METs formed among multiple cells. Using EC Plan-Neofluar 10×/0.30 M27 objective lens wide range of microscopic fields was also captured by the same confocal microscope. The signal of each fluorescence channel was averaged four times. The z-stack images were processed to show maximum-intensity projection. The detailed structure of METs formed by rmCIRP was obtained by a Axio Observer.Z1/7 equipped with Zeiss LSM900 confocal microscopy system. The z-stack images of cells were acquired with Plan-Apochromat 63×/1.40 Oil DIC M27 objective lens. SR-4Y fast acquisition mode of Airyscan and 4× averaging were used. The images obtained by confocal microscope were merged and combined by FIJI ImageJ (22 (link)).
Lsm900 confocal microscopy system
Manufactured by Zeiss
The LSM900 confocal microscopy system is a high-performance imaging solution from Zeiss. It provides advanced optical sectioning and high-resolution imaging capabilities for a range of biological and materials science applications. The system utilizes a laser-scanning approach to capture detailed images of specimens, enabling users to visualize and analyze complex samples with precision.
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3 protocols using lsm900 confocal microscopy system
1
Visualizing the Intricate Structure of Neutrophil Extracellular Traps
2
Visualizing p-MLKL in LPS-induced Cell Death
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RAW cells were cultured on glass bottom petri dishes and treated with LPS and z-VAD-fmk. The nuclei were stained by using NucBlueTM Live cell stain ReadyProbes reagent (Hoechst 33342, Cat. No. 2325910, Invitrogen,) and then rinsed twice with cold PBS. The cell surface membrane was then stained with MemBrite fix Cell Surface staining kit (Biotium, Cat. No. 30093) according to the company’s instructions. Cells were washed with PBS and fixed using 4% methanol for 15 minutes at room temperature. The solution was removed, and the cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour at room temperature. After blocking, cells were incubated in rabbit anti-p-MLKL primary antibody (dilution 1:200; Cat. No. 101375, Cell Signaling Technology) overnight at 4oC. Cells were washed three times and incubated in Alexa Fluor 594 Donkey anti-rabbit IgG (Cat. No. A21207, Thermo Fisher Scientific) for 2 hours. After washing three times, the samples were sealed using prolong gold anti-fade (Cat. No. P36934, Thermo Fisher Scientific). Cells were visualized by using Axio Observer. Z1/7 equipped with Zeiss LSM900 confocal microscopy system. Z-stack images were acquired with Plan-Apochromat 63x/1.40 Oil DIC M27 objective lens. SR-4Y fast acquisition mode of Airyscan and 4× averaging was used. The images obtained by confocal microscope were merged and combined by FIJI Image J.
3
Exosome Uptake in HK-2 Cells
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The PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) was used to label exosomes. Briefly, USC-exosomes were stained with PKH67 dye for 3 min. After centrifugation, the labeled exosomes were resuspended in PBS. The labeled exosomes were added into the culture medium of HK-2 cells. After incubation for 0, 1, 3 h in the dark, the cells were fixed in 4% paraformaldehyde for 20 min and stained with DAPI. Observing exosomes' uptake was visualized using Zeiss LSM900 confocal microscopy system. The images obtained by confocal microscope were merged and combined by FIJI Image J.
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