Lsm 880 confocal laser scanning microscope

Manufactured by Zeiss

Sourced in Germany, United States, United Kingdom, Japan

The ZEISS LSM 880 is a confocal laser scanning microscope. It utilizes laser excitation and a digital detector to capture high-resolution images of samples.

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327 protocols using lsm 880 confocal laser scanning microscope

Visualizing Lipid Droplet and Subcellular Localization

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One milliliter of culture was stained with 10 μL Nile red solution (0.1 mg mL⁻¹ in acetone) and incubated at 37 °C for 30 min in the dark. The stained cells were observed under an LSM880 laser-scanning confocal microscope (Zeiss, Germany) with an excitation wavelength of 514 nm, an emission wavelength of 596 nm, and a detection wavelength range of 539–652 nm.
The subcellular localization of GPAT1 and LPAT1 was determined by fusing EGFP with the 3′-terminus of the target gene in pPhAP1-EGFP following the previous method [30 (link)]. The recombinant plasmids containing the target genes were electroporated into algal cells. The cells were observed under an LSM880 confocal laser scanning microscope (Zeiss, Germany), and the EGFP fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength range of 510–555 nm. Chlorophyll auto-fluorescence was detected at an excitation wavelength of 488 nm and an emission wavelength range of 625–720 nm.

Wang X., Dong H.P., Wei W., Balamurugan S., Yang W.D., Liu J.S, & Li H.Y. (2018). Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum. Biotechnology for Biofuels, 11, 318.

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Subcellular Localization of DHT1 Protein

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For analysis of the subcellular location of the DHT1 protein in plants, the coding region of the DHT1 gene was amplified using primers shown in Supplemental Table S2, and the PCR product was cloned into the Spe I and Bam HI sites of the vector pCAMBIA1305-GFP to generate the plasmid p35S: DHT1-GFP, which was transformed into Kitaake by Agrobacterium-mediated method. GFP fluorescence signal in young roots of transgenic plants was detected with a Zeiss LSM880 confocal laser scanning microscope.
In addition, the full-length coding sequence of DHT1 and truncated cDNA were amplified from Kitaake by PCR using corresponding primers shown in Supplemental Table S2. Subsequently, these fragments were cloned into the Spe I and Xba I sites of the vector PAN580 to generate plasmids that their C-termini were fused to the GFP reporter gene under the control of the CaMV 35S promoter. All of the plasmids were transiently co-expressed into rice protoplasts with the nuclear marker D53-mCherry constructs following the method described previously (Zhang et al., 2011) . The fluorescence signals were observed using a Zeiss LSM880 confocal laser scanning microscope.

Liu T., Zhang X., Zhang H., Cheng Z., Liu J., Zhou C., Luo S., Luo W., Li S., Xing X., Chang Y., Shi C., Ren Y., Zhu S., Lei C., Guo X., Wang J., Zhao Z., Wang H., Zhai H., Lin Q, & Wan J. (2022). Dwarf and High Tillering1 represses rice tillering through mediating the splicing of D14 pre-mRNA. The Plant cell, 34(9).

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Multiplex Immunostaining of FFPE CRC Tissues

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Formalin‐fixed and paraffin‐embedded (FFPE) tissue blocks were chosen from nine CRC patients in Nanfang Hospital for subsequent analysis. Immunostaining was processed with Opal™ 7‐Color IHC Kits (Akoya Biosciences, Marlborough, MA, USA; NEL821001KT), according to the manufacturer's instructions using antibodies (anti‐human CD68 (Proteintech, Chicago, IL, USA; Cat# 66231‐2‐Ig, RRID: AB_2881622); anti‐human APOE (Proteintech; Cat# 66830‐1‐Ig, RRID: AB_2882173); anti‐human CTSZ (Affinity Biosciences, Melbourne, VIC, Australia; Cat# DF14386, RRID:AB_2923130); anti‐human GLUL (Affinity Biosciences; Cat# DF7607, RRID: AB_2841098), and anti‐human FOXP3 (Affinity Biosciences; Cat# AF6544, RRID: AB_2847268)). Images were obtained and analyzed with the Zeiss LSM 880 confocal laser‐scanning microscope (LSM 880; Carl Zeiss, Oberkochen, Germany) and ZEN image analysis software (zen; Carl Zeiss). imagej was used to perform the fluorescence quantitative colocalization analysis [29 (link)].

Wei J., Yu W., Chen J., Huang G., Zhang L., Chen Z., Hu M., Gong X, & Du H. (2023). Single‐cell and spatial analyses reveal the association between gene expression of glutamine synthetase with the immunosuppressive phenotype of APOE+CTSZ+TAM in cancers. Molecular Oncology, 17(4), 611-628.

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Detecting Hrh1 Expression in Mouse Eyes

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Fluorescent in situ hybridization (FISH) was performed by using the RNAscope Multiplex Fluorescent Detection Reagents V2 (Advanced Cell Diagnostics, ACD, Hayward, CA, USA) according to the manufacturer’s instructions. RNAscope probe Mm-Hrh1 (491141) was purchased from ACD. Adult mice were perfused with ice-cold 4% PFA/PBS, and eyes were dissected out and fixed in 4% PFA/PBS at 4 °C overnight. The eyes were dehydrated with increasing concentrations of sucrose solution (10%, 20 and 30%) overnight before embedding in OCT on dry ice. Serial cross sections (12 µm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were pretreated with protease and then subjected to in situ hybridization with RNAscope Multiplex Fluorescent Detection Reagents V2 according to the manufacturer’s instruction (Advanced Cell Diagnostics, Hayward, CA). Briefly, sections were hybridized with the probe solution, followed by amplification and probe detection using TSA plus fluorophores (AKOYA, Marlborough, MA, USA). The sections were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were captured by a Zeiss LSM 880 confocal laser scanning microscope with 40×/1.0 Oil DIC (Carl Zeiss Microscopy, Thornwood, NY, USA).

Chen W., Liu P., Liu D., Huang H., Feng X., Fang F., Li L., Wu J., Liu L., Solow-Cordero D.E, & Hu Y. (2022). Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells. Nature Communications, 13, 6796.

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Monitoring Phagosome pH and Cross-Presentation

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To monitor changes in the phagosome pH via stimulation with the Co1 peptide, sorted cells were co-treated with pHrodo Red Zymosan Bioparticle Conjugate and Alexa 488-conjugated phalloidin (Thermo Fisher Scientific) during stimulation with the Co1 peptide. To test exogenous antigen cross-presentation, sorted cells treated with the OVA + Co1 peptide for 2 h were vigorously washed and further cultured in vitro for 17 h. After fixation, the cells were labeled with APC-conjugated anti-SIINFEKL peptides bound to H-2K^b monoclonal antibodies and Alexa 488-conjugated phalloidin and counterstained with Hoechst 33342. The fluorescence intensity was monitored in Airyscan mode using the Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss).

Kim S.H., Shim E.H., Kim D.J, & Jang Y.S. (2023). C5aR+ dendritic cells fine-tune the Peyer’s patch microenvironment to induce antigen-specific CD8+ T cells. NPJ Vaccines, 8, 120.

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Immunofluorescence Microscopy of CBX7

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Cells were fixed in 4% PFA at room temperature for 10 minutes. The samples were then permeabilized/blocked with PBS containing 0.1% Triton X-100 and 2.5% BSA at room temperature for 1 hour. Samples were then incubated with anti-CBX7 (Abcam, Cat# ab21873, 1:100) at 4 °C overnight. The slides were washed three times with PBS containing 0.1% Tween 20 and incubated with appropriate secondary antibodies or phalloidin (Thermo Fisher Scientific, Cat# A12381) at room temperature for 1–2 hours. DAPI was used for nuclear staining. The samples were visualized under a Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

Cho K.W., Andrade M., Zhang Y, & Yoon Y.S. (2020). Mammalian CBX7 isoforms p36 and p22 exhibit differential responses to serum, varying functions for proliferation, and distinct subcellular localization. Scientific Reports, 10, 8061.

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Immunohistochemical Analysis of c-MET Expression

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Tissue sections were deparaffinized and rehydrated, and antigen retrieval was conducted following the manufacturer’s instructions. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min. Slides were then blocked with goat serum, avidin solution and biotin solution. The slides were incubated with rabbit anti-human polyclonal antibodies against c-MET (1:200 dilution, Proteintech) at 4 °C overnight and then probed with AlexaFlour 594-conjugated secondary antibody (Invitrogen) and mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). Images were acquired on a Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss MicroImaging, Inc, Thornwood, NY).

Zhai Y., Wu W., Xi X, & Yu R. (2020). Adipose-Derived Stem Cells Promote Proliferation and Invasion in Cervical Cancer by Targeting the HGF/c-MET Pathway. Cancer Management and Research, 12, 11823-11832.

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Immunofluorescence Analysis of Aortic Tissue

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Slides of aortic tissue were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized in 0.1% PBS-Triton for 30 min. The samples were blocked in 5% BSA/PBS/0.1% Triton-X 100 for 1 h and then incubated with the primary antibodies anti-Kruppel-like factor 4 (Klf4, ab214666), anti-neural cell adhesion molecule (Ncam1, ab28486), anti-Ki67 (ab15580), anti-alpha-smooth muscle actin (ab7817) (all from Abcam, USA), and anti-vitronectin (Vtn, MA5-24083, Invitrogen, USA). After several PBS washes, the slides were incubated for 2 h at room temperature with the secondary antibodies AF647 donkey anti-mouse and AF568 donkey anti-rabbit (Invitrogen, USA). Immunofluorescence was assessed using a Zeiss LSM 880 confocal laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany). The fluorescence values were measured with ImageJ software (developed at the National Institutes of Health). At least 100 cells were analyzed for each group.

Zhang K., Kan H., Mao A., Geng L, & Ma X. (2021). Single-cell analysis of salt-induced hypertensive mouse aortae reveals cellular heterogeneity and state changes. Experimental & Molecular Medicine, 53(12), 1866-1876.

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Lipid Raft Dynamics and TCR Internalization

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CD3⁺ T blasts were treated with CL (100 µM), CS (100 µM), or vehicle for 3 h and cells were rinsed three times with PBS. Cells were stained with TCRβ-Fab-TRITC for 1 h at 4°C or 37°C. TCRβ internalization was observed by using a 100× NA 1.40 oil immersion objective and an FV1000 laser scanning confocal microscope (Olympus). To determine the influence of CL or CS in lipid raft distribution, cells in each condition were stained with cholera toxin subunit B (CTB)-Alexa594 for 30 min at 37°C. To measure the CL level in the cell membrane, cells in each condition were fixed with 4% paraformaldehyde for 30 min and stained with filipin (50 µg/ml) for 2 h at room temperature (RT). Cells were rinsed with PBS three times and the images were obtained using 405 nm solid-state lasers of Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). Intensity histogram of CTB and filipin were calculated automatically by ZEN software.

Park J.S., Chung I.J., Kim H.R, & Jun C.D. (2023). The Immunosuppressive Potential of Cholesterol Sulfate Through T Cell Microvilli Disruption. Immune Network, 23(3), e29.

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Fixation and Immunofluorescence of BSCs

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BSCs were washed in PBS and then fixed on their inserts in 4% paraformaldehyde (PFA) for 1 h^{56 (link)}. Individual BSCs (n = 1) were cut out from their membranes after fixation and then used as free-floating sections for subsequent steps. BSCs were permeabilized and blocked for 18 h in 0.5% Triton X-100 in 20% bovine serum albumin (BSA) (Sigma Aldrich) at 4 °C. BSCs were then incubated overnight at 4 °C with the appropriate primary antibodies in 5% BSA, washed and then incubated with fluorophore-coupled secondary antibodies for 4 h at RT. Slice cultures were washed a further three times before mounting on slides with Fluoromount-G (Southern Biotech, Birmingham, AL, USA) and then imaged using an Olympus FV1200 IX83 confocal laser-scanning microscope (Olympus America Inc, Center Valley, PA, USA) or Zeiss LSM880 confocal laser-scanning microscope (Carl Zeiss AG, Oberkochen, Germany). Z-stacks were captured at recommended step-sizes and projected as a maximum projection image using the Olympus Fluoview FV10-ASW Version 4.02 or Zeiss Zen Blue 3.4 software.

Croft C.L., Paterno G., Vause A.R., Rowe L.A., Ryu D.H., Goodwin M.S., Moran C.A., Cruz P.E., Giasson B.I, & Golde T.E. (2022). Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance. NPJ Parkinson's Disease, 8, 173.

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