Confocal microscope

Manufactured by Leica Microsystems

Sourced in Germany, United States, Japan

The Leica Confocal Microscope is an advanced imaging instrument that uses a focused laser beam to scan specimens and create high-resolution, three-dimensional images. It enables the study of fine details within samples by optically sectioning through them, providing a clear, in-focus view of specific layers or depths.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (5 mentions) Triton x 100, by Merck Group (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Sp 6000, by Vector Laboratories (2 mentions) Anti p α synuclein, by Abcam (2 mentions) Mouse irdy594, by LI COR (2 mentions) Rabbit irdy488, by LI COR (2 mentions) Vectashield, by Vector Laboratories (2 mentions) Fluoromount g, by Southern Biotech (2 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Alexa 488, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Mitotracker red, by Thermo Fisher Scientific (1 mentions) Cm h2dcfda, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Enzo Life Sciences (1 mentions) Fish kit, by GenePharma (1 mentions)

121 protocols using confocal microscope

Immunohistochemical Analysis of M2 Macrophages

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A 5 μm thick layer of paraffin-coated tissue was cut, decontaminated, and quenched with 3% H₂O₂ for 10 min. To reduce non-specific reactions, the sections were exposed to PBS containing 1% normal human serum, corresponding to the secondary antibody (Thermo Fisher Scientific, USA), and 2% BSA. To analyse M2 macrophages, staining with a monoclonal anti-CD206 and an anti-F4/80 was performed at 4 °C overnight, and then with a corresponding fluorescent secondary antibody. After washing, the slides were treated with DAPI (Sigma-Aldrich) for 5 min at room temperature to stain the nuclei, and the stained cells were examined under Leica Microsystems confocal microscope.

Ding Y., Yang P., Li S., Zhang H., Ding X, & Tan Q. (2022). Resveratrol accelerates wound healing by inducing M2 macrophage polarisation in diabetic mice. Pharmaceutical Biology, 60(1), 2328-2337.

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Visualizing CcNHX1 Localization in N. benthamiana

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Confocal microscopy was utilized to examine the expression and localization of CcNHX1, which was fused with enhanced green fluorescent protein (egfp) coding sequence (CcNHX1-egfp) and driven by the 35S promoter. The binary plasmid construct containing CcNHX1-egfp was introduced into A. tumefaciens strain EHA105. A single colony of EHA105 containing the construct was cultured in Luria-Bertani (LB) broth supplemented with 25 mg/L rifampicin and 100 mg/L kanamycin. After overnight incubation at 28°C, the bacterial culture was centrifuged, and the pellet was resuspended in an infiltration buffer containing 2-(N-morpholino) ethanesulfonic acid (MES) and MgCl₂ at 10 mM; pH 5.85, supplemented with 200 μm acetosyringone. The bacterial suspension was incubated at the room temperature for 4 h, then infiltrated into the fully expanded leaves of Nicotiana benthamiana plants using a 1 mL needleless syringe [45 (link)]. Confocal imaging was performed on samples obtained three days after infiltration using a Leica Microsystems confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).

Mahmoud L.M., Killiny N, & Dutt M. (2024). Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri). PLOS ONE, 19(3), e0294318.

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Immunofluorescence Analysis of Cellular Signaling

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Cells were seeded at 3000 cells/cm2 in confocal culture plates and incubated overnight at 37°C with 5% CO₂. Cultured medium was removed, then added DMEM without FBS for control and Fucoidan-Sargassum solution for treatment, then incubated overnight. Cells were first gently washed with PBS and fixed it using 4% paraformaldehyde solution for 10 min, then washed with PBS for 2 min, permeabilized by Saponin for 8 min, washed with PBS for 3 times 5 min each. Unspecific sites were blocked with 5% goat serum in TBS for 30 min at room temperature. Cells were then labeled for overnight at 4°C with, anti-c-Src (1:100), anti-ITGαV (1:100), anti-SPARC (1:50), anti-GRP94 (1:50), and/or with Alexa-488-conjugated cortactin (1:100), and covered in the dark. After three washes with PBS-T, cells were incubated with FITC-conjugated goat anti-rabbit (1:100), Alexa-647-conjugated goat anti-mouse and Alexa-647-conjugated goat anti-rabbit (1:1000) for 1 h, washed three times with PBS-T. Next, labeled with TRITC-Phalloidin (1:300) for 30min at room temperature, washed three times with PBS-T. Afterward, labeled with DAPI for 3 mins, washed 3 times with PBS-T. Lastly, added antifade mounting medium, the images were taken using Leica Microsystems Confocal Microscope (Wentzler, Germany).

Pan T.J., Li L.X., Zhang J.W., Yang Z.S., Shi D.M., Yang Y.K, & Wu W.Z. (2019). Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVβ3 and Mediating αVβ3/Src/E2F1 Signaling. Journal of Cancer, 10(20), 4777-4792.

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Quantifying Mitochondrial Dynamics in Macrophages

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Intracellular ROS was detected with the specific fluorescent probe CM-H₂DCFDA (Molecular Probes). BMDMs were primed with 200 ng/ml LPS for 2.5 hours, then loaded with 10 μM CM-H₂DCFDA at 37°C for 30 minutes, exposed to 10 μM nigericin and further incubated for 30 minutes. After washing twice with PBS, the cells were analyzed using LSRII (BD Biosciences). For mitochondria morphology, BMDMs were stained for 30 min with 250 nM Mitotracker Red (Invitrogen) and DAPI (Enzo Life Science). Images were captured using a Leica Microsystems confocal microscope with a 63X objective. For the quantification of mitochondrial surface and volume, the 3D Object counter plugin for the Fiji software (version 2.0) was used (18 (link)). Briefly, we used the distance in pixels, the physical distance of the image, and a pixel aspect ratio of 1 to define the pixel size in μm. After defining the threshold between signal and background, the volume and surface of the mitotracker channel is calculated through the image slices.

Moriwaki K., Luz N.F., Balaji S., De Rosa M.J., O’Donnell C.L., Gough P.J., Bertin J., Welsh R.M, & Chan F.K. (2015). The mitochondrial phosphatase PGAM5 is dispensable for necroptosis but promotes inflammasome activation in macrophages. Journal of immunology (Baltimore, Md. : 1950), 196(1), 407-415.

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Detecting circ-TNPO3 localization in ccRCC cells

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The Cy3‐labelled circ‐TNPO3 probes targeting the circ‐TNPO3 back splice site were synthesized at GenePharma (Shanghai, China). ccRCC cells were seeded on a culture plate (iBiDi, Martin Reid, Germany). Localization of circ‐TNPO3 in ccRCC cells was detected by RNA‐fluorescence in situ hybridization (FISH) assay using the FISH kit (GenePharma, Shanghai, China). Images were acquired with a Confocal Microscope (Leica Microsystems, Germany). The information on probes is presented in Table S4.

Pan X., Huang B., Ma Q., Ren J., Liu Y., Wang C., Zhang D., Fu J., Ran L., Yu T., Li H., Wang X., Yang F., Liang C., Zhang Y., Wang S., Ren J., Li W., Wang Y, & Xiao B. (2022). Circular RNA circ‐TNPO3 inhibits clear cell renal cell carcinoma metastasis by binding to IGF2BP2 and destabilizing SERPINH1 mRNA. Clinical and Translational Medicine, 12(7), e994.

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Immunofluorescence Assay for ΔNP63 in T24 Cells

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For IF assay, T24 cells were cultured in 24-well plate. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min at RT, and then washed in PBS for 3 × 3 min. Next, the cells were permeabilized and blocked using 5% BSA (Sigma) and 0.5% Triton X-100 in PBS (PBST) for 1 h at RT, and then incubated with rabbit anti-△NP63 (E6Q3O, CST, USA) in 5% BSA overnight at 4 °C. After removing primary antibody, the cells were washed in PBST for 3 × 3 min, and then incubated in secondary fluorescent antibody (488 nm, Thermo Fisher Scientific, MA, USA) in 5% BSA in a light-proof environment for 1 h at RT. Next, the cells were stained with DAPI (Beyotime, Shanghai, China) in a light-proof environment for 10 min at RT. Finally, the cells were washed with PBS (3 × 5 min, RT) and visualized using a confocal microscope (Leica Microsystems, Mannheim, Germany).

Liu T., Lu Q., Liu J., Xie S., Feng B., Zhu W., Liu M., Liu Y., Zhou X., Sun W., Zhang Y., Chen X., Fang F., Guo H, & Yang R. (2020). Circular RNA FAM114A2 suppresses progression of bladder cancer via regulating ∆NP63 by sponging miR-762. Cell Death & Disease, 11(1), 47.

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Immunohistochemical Analysis of Synovial Tissues

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Synovial tissues were fixed in 10% formalin for about 2 weeks, and embedded in paraffin. Staining methods were previously described in detail^{30 (link)}. Briefly, tissue slides (5 μm thick) were deparaffinized, dehydrated, incubated with proteinase K (Abcam, ab64220), permeablized with TBS-T (0.3% Triton X-100), and eliminated endogenous peroxidase with BLOXALL (Vector Lab, SP-6000). They were then incubated for overnight at 4 °C with the appropriate primary antibodies in antibody diluent (DAKO, S3022). For immunofluorescence, the incubated slide was visualized using secondary antibodies: alexa 488-conjugated anti-mouse antibody (Invitrogen, A11001) and Cy3-conjugated anti-rabbit antibody (Jackson Immunoresearch, 111-165-144). Nuclei were counterstained with DAPI (Thermo Scientific, p36935). To visualize stained cells, confocal microscope (Leica Microsystems, Wetzlar, Germany) was used. For immunohistochemistry, the incubated slide was followed by ABC kit components (Vector Lab, PK-6102), DAB substrate kit (Vector Lab, sk4100), counterstaining with hematoxylin (Merck, 1.05174.0500), and mounting with Permanent mounting medium (Vector Lab, H-5000). To visualize stained cells, images were collected with a Nikon eclipse Ti-U microscope.

Kim Y.R., Kim J.S., Gu S.J., Jo S., Kim S., Young Kim S., Lee D., Jang K., Choo H., Kim T.H., Jung J.U., Min S.J, & Yang C.S. (2020). Identification of highly potent and selective inhibitor, TIPTP, of the p22phox-Rubicon axis as a therapeutic agent for rheumatoid arthritis. Scientific Reports, 10, 4570.

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Cartilage Surface Bioconjugate Imaging

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Following tribological testing, the cartilage surface (~1 mm thick) was sliced off the osteochondral plugs, rinsed three times with PBS, fixed with 4% paraformaldehyde overnight at 4ᵒC, and rinsed three times with PBS. A midsagittal cut was made through the cartilage samples; the two halves were embedded in O.C.T compound (Tissue Tek) and frozen at −80ᵒC. The frozen samples were sectioned at 7 μm thickness, air dried, and stored at −20ᵒC for immunostaining. For detection of biotin-labeled bioconjugates at the cartilage surface, the sections were rinsed with PBS to remove residual O.C.T. compound and blocked with 1% BSA (lyophilized powder, ≥96% (agarose gel electrophoresis), Sigma-Aldrich) for 30 minutes at room temperature. The sections were immunostained for 15 minutes at room temperature with Alexa Fluor® Streptavidin 633 (Thermo Scientific) diluted 1:200 in 1% BSA and counterstained with DAPI diluted 1:500 in the same solution. The sections were rinsed and mounted with ProLong Gold antifade mounting medium (Thermo Scientific) before they were imaged (CS control at 488 and all other surfaces at 633) under a confocal microscope (Leica Microsystems, Buffalo Grove, IL). Chondroitin sulfate-Alexa Fluor® 488 (Thermo Scientific) conjugates with no biotinylated peptides were analyzed as a control.

Twitchell C., Walimbe T., Liu J.C, & Panitch A. (2020). PEPTIDE-MODIFIED CHONDROITIN SULFATE REDUCES COEFFICIENT OF FRICTION AT ARTICULAR CARTILAGE SURFACE. Current research in biotechnology, 2, 16-21.

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Immunofluorescence Analysis of Osteogenic Markers

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The differentiated osteoprogenitors were washed twice with PBS and fixed with 10% formalin for 15 min. This was followed by permeabilization with PBS containing 0.1% Triton X-100 and 1% BSA for 1 h and incubation with primary antibody overnight. Then, they were washed with PBS and incubated with Cy3 or Alexa 488-conjugated secondary antibody for 1 h. The stained cells were washed with distilled water and mounted with DAPI (H1200, Vector, Burlingame, CA, USA). To visualize stained cells, immunofluorescence images were obtained using a confocal microscope (Leica Microsystems, Wetzlar, Germany). Antibodies used in IF were as follows: ON (sc-73472; Santa Cruz Biotechnology, Dallas, TX, USA), OCN (sc-365797; Santa Cruz, TX, USA), Alexa-488 (A11001; Thermo Fisher), and Cy3 (A10520, Thermo Fisher).

Nam B., Park H., Lee Y.L., Oh Y., Park J., Kim S.Y., Weon S., Choi S.H., Yang J.H., Jo S, & Kim T.H. (2020). TGFβ1 Suppressed Matrix Mineralization of Osteoblasts Differentiation by Regulating SMURF1–C/EBPβ–DKK1 Axis. International Journal of Molecular Sciences, 21(24), 9771.

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Immunofluorescence Analysis of AKT2 Expression

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Cells were fixed with 4% paraformaldehyde and pre-incubated with 10% normal goat serum (KPL) at room temperature and then incubated with primary antibody anti-AKT2 (mouse monoclonal, 1:100, ab175354, Abcam) at 4°C overnight. Secondary antibody was FITC-labeled antibody to mouse IgG (cat. no. 5230-0307, KPL, 1:50, US) and incubated at room temperature for 2 h. Antifading mounting medium (with DAPI) (Beijing Solarbio Science & Technology Co., Ltd.) was used for nuclear staining at room temperature and stored at 4°C. Images were captured using a confocal microscope (Leica Microsystems GmbH) and digitized using LAS AF software (v. 2.6.0, Leica Microsystems GmbH).

Wang S., Zheng B., Zhao H., Li Y., Zhang X, & Wen J. (2021). Downregulation of lncRNA MIR181A2HG by high glucose impairs vascular endothelial cell proliferation and migration through the dysregulation of the miRNAs/AKT2 axis. International Journal of Molecular Medicine, 47(4), 35.

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