Lsm 880 confocal microscope

Manufactured by Zeiss

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The ZEISS LSM 880 is a confocal microscope that allows for high-resolution imaging of biological samples. It features advanced optics and advanced detectors to provide detailed, three-dimensional visualization of specimens.

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1 702 protocols using lsm 880 confocal microscope

Subcellular Localization of RCL1 Protein

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To determine the subcellular localization of RCL1, we generated two constructs: p35S-RCL1-GFP (full-length CDs of RCL1) and p35S-RRP7-RFP (full-length CDs of AtRRP7). The fragments were amplified from cDNA prepared from 10 DAP B73 endosperm and A. thaliana leaves. The resulting PCR products were cloned into the BamH I and Sal I (RCL1) sites of pCAMBIA1300-35S-GFP, or Kpn I and Xba I (AtRRP7) sites of pCAMBIA1300-35S-RFP to create fusion proteins. The plasmids were transferred into Agrobacterium tumefaciens (GV3101 strain) and injected into 3-week-old N. benthamiana leaves. Two days later, the injected areas were observed using an LSM880 confocal microscope (Zeiss, Jena, Germany). For ZmRCL1 subcellular localization in maize, the constructs of 35S-RCL1-GFP and 35S-RRP7-RFP were co-transformed into maize endosperm protoplasts using polyethylene glycol-mediated transformation as previously described (Yoo et al., 2007 (link)). The B73 endosperm protoplasts were isolated at eight DAP. The transformed protoplasts were cultured at 25°C in the dark for 36 h, and fluorescence was observed via confocal microscopy using an LSM880 confocal microscope (Zeiss, Jena, Germany).

Wang T., Chang Y., Zhao K., Dong Q, & Yang J. (2022). Maize RNA 3'-terminal phosphate cyclase-like protein promotes 18S pre-rRNA cleavage and is important for kernel development. The Plant Cell, 34(5), 1957-1979.

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Protein Localization in Meiotic Flower Buds

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For protein localization experiments, healthy flower buds were dissected exposing two anthers and carefully positioned in a petri plate filled with half-strength MS medium, pH 5.8 solidified with 0.8% (w/v) agar, and meiocytes of different meiotic stages were imaged using a Zeiss LSM880 confocal microscope.
For pixel intensity plots, flower buds were dissected and the anthers in MT array state 6 were imaged using a Zeiss LSM880 confocal microscope with the exact same settings for the different heat conditions. The pixel brightness was measured through a region of interest using ImageJ (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)) and plotted against the X dimension, which is the distance of the region of interest.

De Jaeger-Braet J., Krause L., Buchholz A, & Schnittger A. (2021). Heat stress reveals a specialized variant of the pachytene checkpoint in meiosis of Arabidopsis thaliana. The Plant Cell, 34(1), 433-454.

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Visualizing Macrophage Phagocytosis of Apoptotic Endothelial Cells

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To identify macrophage phagocytosis of apoECs, THP1 cells that were exposed to apoptotic GFP-hECs for 24 h on a chamber slide were fixed in 4% paraformaldehyde for 10 minutes, and then permeabilized by incubation with 0.15% Triton X 100 for 10 minutes. The cells were then treated with anti-CD44-APC (1:50) for 1 hour. Fluorescent images were recorded after washing with PBS using a LSM 880 confocal microscope (ZEISS, Oberkochen, Germany). To identify macrophage phagocytosis of apoECs in skin wounds, wound tissues were embedded in frozen Tissue-Plus Optimal Cutting Temperature Compound (Fisher Scientific, Hampton, NH) and cut into 8μM sections for fluorescent microscopy or 12 μM sections for confocal imaging. Sections were blocked with 10% goat serum in PBS for 45 min and then incubated with rat anti-mouse CD68 (Bio-Rad Laboratories, Hercules, CA, dilution 1:1000) for 45 min. After washing with PBS, the sections were incubated with an Alexa fluor 594 goat anti-rat IgG (Invitrogen, Carlsbad, CA, dilution 1:1000). All procedures were performed at room temperature. Fluorescent images were acquired using a Zeiss fluorescence microscope, Axioskop 40, or LSM 880 confocal microscope (ZEISS) and analyzed using Zeiss Zen Blue software (ZEISS).

Xu M., Chen Z., Chen K., Ma D., Chen L, & DiPietro L.A. (2021). Phagocytosis of apoptotic endothelial cells reprograms macrophages in skin wounds. Journal of immunology and regenerative medicine, 12, 100038.

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Measuring Lysosomal Calcium Release

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Cells were grown on 15 mm coverslips and transfected with a plasmid encoding a perilysosomal GCaMP3-ML1 Ca²⁺ probe. After 48 h, cells were stained with lysotracker and lysosomal Ca²⁺ release was measured in a basal Ca²⁺ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA, and 20 mM HEPES (pH 7.4) with or without Kaem, by monitoring fluorescence intensity at 470 nm with an LSM880 confocal microscope (Zeiss). For glycyl-l-phenylalanine-β-naphthylamide (GPN) pretreatment experiment, 48 h transfection with GCaMP3-ML1, cells were trypsinized and plated onto a glass-bottom plate. The experiment was carried out 3–5 h after plating when cells still exhibited round morphology. lysosomal Ca²⁺ release was measured in a basal Ca²⁺ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA, and 20 mM HEPES (pH 7.4) with or without GPN (100 μM) pretreatment, by monitoring fluorescence intensity at 470 nm with an LSM880 confocal microscope (Zeiss).

Kim D., Hwang H.Y., Ji E.S., Kim J.Y., Yoo J.S, & Kwon H.J. (2021). Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction. Communications Biology, 4, 1.

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Apoptosis Detection Protocols

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TUNEL detection kit (G002) purchased from Nanjing Jiancheng Bioengineering Institute and mitochondrial membrane potential assay kit with JC-1 (C2006) from Beyotime Biotechnology were used for cell apoptosis detection. All procedures were performed based on the instruction from the kits. Briefly, the cells with different treatments were incubated with JC-1 staining solution at 37°C for 20 minutes and then washed twice with JC-1 staining buffer before being observed under an LSM-880 confocal microscope (Carl Zeiss, Oberkochen, Germany). For terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, the cells after various treatments were fixed with 4% paraformaldehyde for 30 min and then incubated with 1% BSA for 10 min. After that, the samples were incubated with 0.5% Triton X-100 for 20 min, which was followed by TdT enzyme reaction solution for 60 min treatment. Finally, DAPI was used for nucleus staining for 5 min before observing with an LSM-880 confocal microscope (Carl Zeiss, Oberkochen, Germany). And the quantitative result of TUNEL-positive cells and the fluorescence ratio of red and green were carried out through ImageJ.

Xiang C., Zhang F., Gao J., Guo F., Zhang M., Zhou R., Wei J., Wang P., Zhang Y., Zhang J, & Yang H. (2021). Yixin-Shu Capsules Ameliorated Ischemia-Induced Heart Failure by Restoring Trx2 and Inhibiting JNK/p38 Activation. Oxidative Medicine and Cellular Longevity, 2021, 8049079.

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Visualizing Microtubules in Guard Cells

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Microtubules in guard cells were visualized by confocal microscopy using a 100× oil objective lens (Zeiss LSM 880 confocal microscope). The detached leaves were incubated in opening solution (MES buffer, as described above) under constant light for 3 h to open the stomata completely, followed by treatment with MES buffer containing different drugs for the indicated times. The cortical microtubules were observed by detecting the YFP fluorescence signal under a Zeiss LSM 880 confocal microscope equipped with a 488-nm argon ion laser as the excitation source and a 505-to 550-nm band-pass filter, and mCherry was excited at 561 nm. Experiments were repeated at least three times on a minimum of 15 guard cells.

Wang P., Qi S., Wang X., Dou L., Jia M.A., Mao T., Guo Y, & Wang X. (2023). The OPEN STOMATA1-SPIRAL1 module regulates microtubule stability during abscisic acid-induced stomatal closure in Arabidopsis. The Plant cell, 35(1).

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Immunofluorescence and Aggregate Analysis

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Cells grown on coverslips were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed in 0.1% glycine in PBS for 5 min, permeabilised in 0.1% Triton X-100 in PBS for 10 min. 1% BSA in PBS was used for blocking (20 min), primary antibody incubation (1 h) and secondary antibody incubation (20 min). The experiments visualising mRFP-GFP-LC3 were fixed with 4% PFA for 5 min. Coverslips were mounted with ProLong Gold Antifade Reagent (Molecular Probes). A Zeiss LSM880 confocal microscope was used for fluorescent confocal analysis. All confocal images were taken with a 63x oil-immersion lens. EGFP-HTT(Q74) aggregation was monitored with a fluorescence microscope using a 100x oil-immersion lens. At least 250 cells were counted per coverslip and the proportion of cells with at least one aggregate was scored as a percentage of the total number of transfected cells. A Zeiss LSM880 confocal microscope for representative imaging with a 40x oil-immersion lens.

Karabiyik C., Vicinanza M., Son S.M, & Rubinsztein D.C. (2021). Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner. Developmental cell, 56(13).

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Spatial Expression of AlinOBP14 in Acalymma

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After 12 h, the expression of the AlinOBP14 gene in antennae, stylets, the heads without antennae and stylets, thoraces, abdomens, legs, and wings from injected and non-injected A. lineolatus adults of both genders was observed by a Zeiss LSM 880 confocal microscope. In brief, the different tissues of injected and non-injected A. lineolatus were embedded in Tissue-Tek optimal cutting temperature (O.C.T.) compound (Sakura Finetek, Torrance, CA, USA) and cut into 12 µm slices at −20 °C by using a freezing microtome (Thermo scientific, Cryostar NX50, Waltham, MA, USA). Sections were collected on superfrost plus microscope slides (Fisher Scientific, USA). The slides were dried at room temperature and fixed in 4% paraformaldehyde solution at 4 °C for 30 min, incubated in 0.2 M HCl for 10 min, and washed in PBS buffer. The slides were mounted using ProLong^TM Glass antifade mountant (P36980, Thermo Fisher Scientific, Waltham, MA, USA). The tissues were observed using a Zeiss LSM880 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Images and colocation characteristics of enhanced fluorescent signals were processed with ZEN 3.2 (Carl Zeiss Microscopy GmbH, Jena, Germany).

Tian W., Zhang T., Gu S., Guo Y., Gao X, & Zhang Y. (2021). OBP14 (Odorant-Binding Protein) Sensing in Adelphocoris lineolatus Based on Peptide Nucleic Acid and Graphene Oxide. Insects, 12(5), 422.

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Visualizing Intracellular Preribosomes and AFs

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The intracellular location of preribosomes or AFs was determined as follows: 35 mm glass bottom microwell dishes (MaTek) were coated with concanavalin A (Sigma) and allowed to dry for 40 min. Cells expressing GFP-tagged uL23 or Arx1-GFP were grown to an OD₆₀₀ of 0.3–0.5 before plating 10 μL onto the glass bottom of the MaTek dish and incubating at room temperature for 20 min. Plates were then washed once with 1 mL of appropriate media before being overlaid with 2 mL of media and then imaged using a Ziess LSM 880 confocal microscope. Images were processed using Fiji⁴⁶.

Wilson D.M., Li Y., LaPeruta A., Gamalinda M., Gao N, & Woolford JL J.r. (2020). Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel. Nature Communications, 11, 5111.

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Immunocytochemistry of Adhesion Proteins

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The following primary antibodies were used for immunocytochemistry; mouse anti-Vinculin (1:500, Sigma Aldrich V9131), rabbit anti-Paxillin [Y113] (1:500, Abcam ab32084), rabbit anti-β3/CD61 [SJ19-09] (1:500, Invitrogen MA5-32077), and rat anti-β1 [MB1.2] (1:250, Sigma MAB1997) .
For confocal microscopy, cells were prepared as described above. Following fixation and permeabilization, coverslips were washed three times with PBS and then blocked for 30 minutes with 1% Bovine Serum Albumin (BSA, Sigma) in PBS + 0.2% Tween20 (PBS-T). After blocking, coverslips were incubated with primary antibodies diluted in PBS-T with 1% BSA for 1hr at RT or overnight at 4°C. Coverslips were washed with PBS-T three times for 5 minutes followed by the addition of the appropriate Alexa Fluor 488, 568, and/or 647 conjugated secondary antibodies (1:1000, Invitrogen) or Alexa Fluor 647-Phalloidin (1:400, Invitrogen) diluted in PBS-T with 1% BSA 1 h at RT or overnight at 4 °C. Following this, cells were washed 3 times in PBS-T then incubated with DAPI (Sigma) 1:1000 or Hoechst (Sigma) 1:2000 in PBS for 20 min. Cells were then washed and mounted on glass slides using Thermo Immu-Mount and imaged on a Ziess LSM 880 Confocal Microscope.

Riker K.D., Daly M.L., Papanikolas M.J., Papanikolas M.J., Jian T., Klawa S.J., Shin Sahin J.Y.S., Liu D., Singh A., Miller A.G, & Freeman R. (2021). A Programmable Toolkit to Dynamically Signal Cells Using Peptide Strand Displacement. ACS applied materials & interfaces, 13(18).

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