Sp5 inverted 2 photon flim confocal

Manufactured by Leica

The Leica SP5 Inverted 2-Photon FLIM confocal is a versatile microscope system designed for advanced imaging applications. It combines the capabilities of a confocal microscope with the depth-penetrating power of two-photon excitation and the ability to perform fluorescence lifetime imaging (FLIM). This system is equipped with high-sensitivity detectors and a tunable excitation laser, enabling researchers to capture detailed, high-quality images of samples.

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Inverted Confocal SP5 Confocal SP5-II SP5 Inverted Confocal SP5 SP5 II

5 protocols using sp5 inverted 2 photon flim confocal

Quantitative Analysis of cMet Expression

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The cells were blocked with 1X PBS plus 2% BSA for 1 hour at 4 °C, and were incubated with 5 μM of peptide for 10 min at room temperature (RT) in the dark, washed 3×, and fixed with 4% PFA for 5 min, washed with 1X PBS, and then mounted on glass slides with ProLong Gold reagent containing DAPI (Invitrogen). A 1:3000 dilution of primary monoclonal rabbit anti-cMet antibody (#8198, Cell Signaling Technology) was incubated with the cells in vitro as a positive control. Afterwards, the cells were incubated with a 1:500 dilution of AF488-labeled secondary goat ant-rabbit immunoglobulin G antibody (#A-11029, Life Technologies), and then mounted on glass slides with ProLong Gold reagent containing DAPI. Confocal fluorescence images were collected using a 63X oil-immersion objective (Leica SP5 Inverted 2-Photon FLIM confocal). Fluorescence intensities were quantified using custom Matlab software (Mathworks).

Wu X., Zhou J., Wang F., Meng X., Chen J., Chang T.S., Lee M., Li G., Li X., Appelman H.D., Kuick R, & Wang T.D. (2019). Detection of colonic neoplasia in vivo using near-infrared-labeled peptide targeting cMet. Scientific Reports, 9, 17917.

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Immunofluorescence Imaging of GBP1-5 and HSP90

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A375 cells mounted on cover slips were treated with IFNγ for 24 hours. After washing 2 times with PBS, cells were fixed with 4% PFA for 15 minutes and washed 2 times with PBS for 5 minutes each. Then, the cells were permeabilized with 0.5% triton x-100 in PBS for 10 minutes and rinsed 2 times with PBS for 5 minutes each. Antigens were blocked with 10% normal goal serum in PBS for 30 minutes. Then, primary antibodies were added at 1:50 dilutions of mouse anti-human GBP1-5 antibody (Santa Cruz, 166960) or rabbit anti-human HSP90 antibody (CST, 4877), and incubated at 4°C overnight. Afterwards, the cells were washed and incubated with 1:500 dilutions of Qdot 605 labeled secondary goat anti-mouse antibody (Thermo Fisher, Q11002) or AF488-labeled secondary goat anti-rabbit antibody (Thermo Fisher, A11034), and then mounted on glass slides with ProLong Gold reagent containing DAPI. Confocal fluorescence images were collected using a 63X oil-immersion objective (Leica SP5 Inverted 2-Photon FLIM confocal).

Li G., Kryczek I., Nam J., Li X., Li S., Li J., Wei S., Grove S., Vatan L., Zhou J., Du W., Lin H., Wang T., Subramanian C., Moon J.J., Cieslik M., Cohen M, & Zou W. (2021). LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy. Nature cell biology, 23(5), 526-537.

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Immunofluorescence Microscopy Protocol

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Cells were fixed with 4% PFA in PBS for 10 min at RT and then were permeabilized with 0.1% saponin or 0.05% TritonX-100 in PBS for 10 min at RT, washed twice with PBS, and blocked with PBS containing 0.1% gelatin for 30 min at RT or overnight at 4°C. Cells were incubated with primary antibodies in PBS containing 0.1% gelatin for 60 min. After three washes with PBS, cells were incubated with the appropriate secondary antibodies in PBS containing 0.1% gelatin for 50 min. After a brief wash with PBS, coverslips were mounted onto slides using ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific) and observed under a Leica SP5 Inverted 2-Photon FLIM Confocal equipped with 63×/NA 1.40 oil immersion objective lens (Leica).

Hasegawa J., Tokuda E., Yao Y., Sasaki T., Inoki K, & Weisman L.S. (2022). PP2A-dependent TFEB activation is blocked by PIKfyve-induced mTORC1 activity. Molecular Biology of the Cell, 33(3), ar26.

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Immunofluorescence Imaging of GBP1-5 and HSP90

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Visualizing Intestinal Tight Junctions

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In all, 8-μm OCT-embedded tissue sections of mouse ileum were fixed and permeabilized in absolute ethanol at −20 °C for 20 min, blocked with 3% donkey serum-PBS for 1 h at room temperature (RT) and incubated with primary antibody goat anti-mouse JAM-A (R&D systems) diluted in blocking buffer overnight at 4 °C. Alexa Fluor 488 donkey anti-goat antibody (Life technologies) diluted in blocking buffer was added for 1 h at RT. To visualize cell nuclei, tissue sections were incubated with Dapi (Life technologies) for 10 min at RT then mounted in Prolong Antifade Agent (Life technologies). Immunofluorescence images were captured on a confocal microscope (Leica SP5 Inverted 2-Photon FLIM Confocal). ImageJ (National Institutes of Health, Bethesda, MD) was used for image processing.

Luissint A.C., Williams H.C., Kim W., Flemming S., Azcutia V., Hilgarth R.S., O’ Leary M.N., Denning T.L., Nusrat A, & Parkos C.A. (2019). Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice. Mucosal immunology, 12(3), 668-678.

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