Lsm 710 confocal microscope system

Manufactured by Zeiss

Sourced in Germany

The LSM 710 is a confocal microscope system manufactured by Zeiss. It is designed for high-resolution imaging of biological samples. The system utilizes laser excitation and a pinhole aperture to achieve optical sectioning, enabling the capture of detailed 3D images.

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51 protocols using lsm 710 confocal microscope system

Monitoring Autophagic Flux with tfLC3

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To monitor the autophagic flux, we used a tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) reporter to as previously described.²⁵ To evaluate tandem fluorescent LC3 puncta, cells were transfected with tfLC3 alone or co‐transfected with siRNA (anti‐Beclin‐1 or non‐targeting siRNA) and tfLC3. 48 hours post‐transfection, the cells were washed with 1X PBS and immediately analysed via confocal microscopy using a Zeiss LSM 710 confocal microscope system (Carl Zeiss). Images analysis was performed by the ZENLE confocal analysis software (Carl Zeiss).

Huang M., Qi C., Zou Y., Yang R., Jiang Y., Sheng J., Kong Y., Tao Z, & Chen S. (2020). Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway. Journal of Cellular and Molecular Medicine, 24(14), 7778-7788.

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Multi-modal Microscopy Techniques

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All fluorescent and bright-field microscopy–based assays were observed using a Nikon Eclipse Ti microscope. High-resolution confocal fluorescent microscopy was performed using a Zeiss LSM 710 confocal microscope system (Carl Zeiss Inc.) and visualized using the ZEN Zeiss Imaging software. Electron microscopy was carried out as previously described (24310399) using transmission electron microscopy Tecnai G² Spirit BioTWIN with an AMT 2k CCD camera.

Ricklefs F.L., Alayo Q., Krenzlin H., Mahmoud A.B., Speranza M.C., Nakashima H., Hayes J.L., Lee K., Balaj L., Passaro C., Rooj A.K., Krasemann S., Carter B.S., Chen C.C., Steed T., Treiber J., Rodig S., Yang K., Nakano I., Lee H., Weissleder R., Breakefield X.O., Godlewski J., Westphal M., Lamszus K., Freeman G.J., Bronisz A., Lawler S.E, & Chiocca E.A. (2018). Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Science Advances, 4(3), eaar2766.

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Proximity Ligation Assay for Protein Interactions

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The proximity ligation assay (PLA) analysis was conducted in accordance with previously established protocols⁴⁰ (link). HEK293T cells were cultured on a Biosharp confocal dish for no less than 18 h before being subjected to two rounds of PBS washing and fixed in 4% formaldehyde PBS solution at room temperature for 15 min. The resulting samples were then rinsed with TBST. The dishes were subsequently blocked with a BSA blocking solution at 37 °C for 1.5 h, followed by 24 h incubation with antibody combinations at 4 °C. After washing with TBST, proximity ligation was performed using Rabbit PLUS and Mouse MINUS Duolink in situ PLA kits (Sigma–Aldrich) according to the manufacturer's instructions. The dishes were subsequently immersed in DAPI solution for nuclear restaining. The images were analyzed using Zeiss confocal software and captured by the Zeiss LSM710 confocal microscope system (Carl Zeiss GmbH, Jena, Germany).

Cheng C., Yao H., Li H., Liu J., Liu Z., Wu Y., Zhu L., Hu H., Fang Z, & Wu L. (2024). Blockade of the deubiquitinating enzyme USP48 degrades oncogenic HMGA2 and inhibits colorectal cancer invasion and metastasis. Acta Pharmaceutica Sinica. B, 14(4), 1624-1643.

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Monitoring Autophagic Flux with tfLC3

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Tandem monomeric RFP-GFP-tagged LC3 (tfLC3) (HB-AP2100001, Hanbio, China) was used to monitor autophagic flux as previously reported. LC3-II relocalized to the autophagosomal membranes during autophagy. Thus, the accumulation of mRFP-GFP-LC3 puncta is an effective way to detect autophagosomes. When tfLC3 is located in autolysosomes, this form of LC3 displays only red fluorescence since the GFP signal is sensitive to the acidic condition in the lysosome lumen, whereas the RFP signal is more stable. To evaluate tandem fluorescent LC3 puncta, 48 h after tfLC3 transfection, cells were washed once with 1 × PBS, incubated with EBSS (E2888, Sigma) for the indicated durations and then directly sent out for confocal microscopy analysis. Images of samples were captured using a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Germany) and processed with ZEN LE software (Carl Zeiss).

Xu S., Wang P., Zhang J., Wu H., Sui S., Zhang J., Wang Q., Qiao K., Yang W., Xu H, & Pang D. (2019). Ai-lncRNA EGOT enhancing autophagy sensitizes paclitaxel cytotoxicity via upregulation of ITPR1 expression by RNA-RNA and RNA-protein interactions in human cancer. Molecular Cancer, 18, 89.

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Western Blot and Immunofluorescence Analysis

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Cells were washed with phosphate buffered solution (PBS) and whole cell lysate was prepared with modified RIPA buffer. Supernatants of the homogenates were subjected to 4%–12% Bis-Tris gel by electrophoresis, and transferred to PVDF membranes. The membranes were probed with anti-vimentin, anti-GAPDH, anti-rabbit or mouse IgG horseradish peroxidase (Sigma, St. Louis, MO, USA), anti-BMI-1 (Millipore Inc., Billerica, MA, USA), anti-AKT, anti-p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GSK3b, anti-p-GSK3b, anti-snail, anti-α-catenin, anti-β-catenin (Cell Signaling Technology, Beverly, CA, USA) and detected with ECL Western blotting detection reagents (Thermo Fisher, Palo Alto, CA, USA).
Immunofluorescence staining was performed on cells plated in chamber slides. The cells were fixed in 3.7% formaldehyde for 15 min, washed three times with PBS and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Mouse monoclonal anti-snail, anti-vimentin, and Alex Fluor®568 goat anti-mouse IgG (Millipore Inc.) were used as primary and secondary antibodies, respectively. Images were obtained using a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Jena, Germany).

Xu Z., Zhou Z., Zhang J., Xuan F., Fan M., Zhou D., Liuyang Z., Ma X., Hong Y., Wang Y., Sharma S., Dong Q, & Wang G. (2020). Targeting BMI-1-mediated epithelial–mesenchymal transition to inhibit colorectal cancer liver metastasis. Acta Pharmaceutica Sinica. B, 11(5), 1274-1285.

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Immunohistochemical Brain Analysis in Mice

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Mice were euthanized using CO₂ inhalation and subsequently perfused with neutral-buffered formalin 4% (Sigma-Aldrich, MO) for fixation. Cryoprotection was performed using 30% sucrose. All mouse brain slides were obtained from frozen sections. Permeabilization was done using 1% Triton-X-100 (Sigma-Aldrich) in PBS pH 7.4 for 10 min. Slides were then incubated with the primary antibody (1:100 in normal serum) overnight at 4°C. For detection of the primary antibody, species-matched fluorophore coupled antibodies were incubated for 1 h at room temperature. Slides were then covered with anti-fade mounting medium (Vectashield, Vectorlabs, CA) and coverslipped. All fluorescent and bright-field microscopy-based assays were observed using a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan). High resolution confocal fluorescent microscopy was performed using a Zeiss LSM 710 confocal microscope system (Carl Zeiss Inc., Germany) and visualized using the ZEN Zeiss Imaging software.

Krenzlin H., Zdioruk M., Nowicki M.O., Finkelberg T., Keric N., Lemmermann N., Skubal M., Chiocca E.A., Cook C.H, & Lawler S.E. (2021). Cytomegalovirus infection of glioblastoma cells leads to NF-κB dependent upregulation of the c-MET oncogenic tyrosine kinase. Cancer letters, 513, 26-35.

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Immunofluorescence Staining of Cellular Proteins

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Cellular adhesive molecules and ECM proteins from collected cells were stained without permeabilization. For intracellular signaling molecules and cytoskeletal protein components, the fixed cells were permeabilized with 0.4% Triton X-100 in DPBS for 15 min and incubated successively in blocking buffer (1% BSA in DPBS), primary antibodies (10 μg/mL in blocking buffer) and secondary antibodies (5 μg/mL in blocking buffer) for 1 h at 37°C. After each step, the cells were washed three times in DPBS. The images of stained cells were collected using a Zeiss LSM710 confocal microscope system (Zeiss, Oberkochen, Germany) with a 63× oil immersion objective.

Li N., Wang C., Sun S., Zhang C., Lü D., Chen Q, & Long M. (2018). Microgravity-Induced Alterations of Inflammation-Related Mechanotransduction in Endothelial Cells on Board SJ-10 Satellite. Frontiers in Physiology, 9, 1025.

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TUNEL Assay for Apoptosis Quantification

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Apoptosis was determined using the TUNEL apoptotic cell detection kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The percentages of apoptotic cells were counted from at least 1000 cells. The confocal images of cells were sequentially acquired with Zeiss AIM software on a Zeiss LSM 710 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany) and images were processed with ZEN LE software (Carl Zeiss).

Xu W., Wei Q., Han M., Zhou B., Wang H., Zhang J., Wang Q., Sun J., Feng L., Wang S., Ye Y., Wang X., Zhou J, & Jin H. (2018). CCL2-SQSTM1 positive feedback loop suppresses autophagy to promote chemoresistance in gastric cancer. International Journal of Biological Sciences, 14(9), 1054-1066.

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Immunocytochemical Analysis of Neuronal Markers

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Neurons were seeded on cover slips pre-coated with 20 μg/ml poly-L-ornithine and 10 μg/ml laminin. Cell monolayers were fixed with 4% paraformaldehyde (Sigma Aldrich) and, after permeabilization with blocking buffer (5% donkey serum and 1% triton in PBS) at RT for 1 h, cells were incubated with primary antibodies diluted in blocking buffer overnight at 4°C. The primary antibodies used were: anti-Synapsin-1 (1:200, #5297; Cell Signaling Technology); anti-MAP2 (1:500, M9942; Sigma-Aldrich). After PBS washings, cells were incubated at RT for 1 h with secondary antibodies conjugated with AlexaFluor594 or AlexaFluor488 (Thermo Fisher Scientific; 1:400). Nuclei were counterstained with DAPI (VECTASHIELD, Vector laboratories). Fluorescence images were obtained using a Zeiss LSM 710 confocal microscope system (Carl Zeiss Meditec AG) and used as an initial qualitative test.

Gomes A.K., Dantas R.M., Yokota B.Y., Silva A.L., Griesi-Oliveira K., Passos-Bueno M.R, & Sertié A.L. (2022). Interleukin-17a Induces Neuronal Differentiation of Induced-Pluripotent Stem Cell-Derived Neural Progenitors From Autistic and Control Subjects. Frontiers in Neuroscience, 16, 828646.

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Lysosomal and Autophagy Dynamics in ARPE-19 Cells

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ARPE-19 cells were cultured on polyethyleneimine - coated coverslips sit in 6-well plates. After treated with LA, Epo, or sham-treatment, the cells were firstly labeled by incubating with lysotracker (Invitrogen), a lysosome reporter dye, for 90 min at 37°C. After washed with PBS, the cells were fixed in 4% paraformaldehyde for 5–10 min, washed in PBS, blocked in goat sera for 45 min, and then incubated with LC3 antibody (1∶250) in 0.1% Triton-X100 for 2 h following incubated with FITC-conjugated goat anti-mouse IgG in 0.1% Triton-X100 for another 45 min. Finally, the nuclei were stained with DAPI for 3 min, washed, and then observed under a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Germany). The images were taken under oil-immersion lens (X 63) and processed with Zen Le software. All the procedures were performed under ambient temperature.

Tang B., Cai J., Sun L., Li Y., Qu J., Snider B.J, & Wu S. (2014). Proteasome Inhibitors Activate Autophagy Involving Inhibition of PI3K-Akt-mTOR Pathway as an Anti-Oxidation Defense in Human RPE Cells. PLoS ONE, 9(7), e103364.

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