Immunofluorescence was performed as described previously5 (link). Briefly, cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were washed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing two times, cells were blocked in 10% BSA in PBS for 1 h at room temperature. Cells were then incubated with primary antibodies in 5% BSA in PBS supplemented with 0.1% Tween 20 (PBST) overnight at 4 °C. The next day, the cells were washed four times with PBST, each for 10 min, followed by incubation with Alexa Fluor-conjugated secondary antibody (Life Technologies), in 5% BSA/PBST for 1 h at room temperature. The cells were then washed four times in PBST, incubated with 1 µg/ml DAPI in PBS for 5 min, and washed twice with PBS. The slides were mounted with ProLong Gold (Life Technologies), and imaged with Leica TCS SP8 fluorescent confocal microscope. Quantification of % positive cells of SAHF, p-ATM, IL8, γH2AX, and p65 was done under identical microscopy settings between samples. Cells with over 5 visible spots at the expected location were considered positive. Over 200 cells from 4 randomly-selected fields were analyzed.
Tcs sp8 fluorescent confocal microscope
Manufactured by Leica
The Leica TCS SP8 is a fluorescent confocal microscope. It is designed for high-resolution imaging of fluorescently labeled samples. The system combines a confocal laser scanning microscope with a sensitive detection system to provide detailed visualization of cellular and subcellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (4 mentions)
Prolong gold, by Thermo Fisher Scientific (4 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Anti mouse cd3e, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd28, by Thermo Fisher Scientific (1 mentions)
6 protocols using tcs sp8 fluorescent confocal microscope
1
Immunofluorescence Assay for Nuclear Markers
2
Immunofluorescence Assay for Nuclear Markers
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Immunofluorescence was performed as described previously5 (link). Briefly, cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were washed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing two times, cells were blocked in 10% BSA in PBS for 1 h at room temperature. Cells were then incubated with primary antibodies in 5% BSA in PBS supplemented with 0.1% Tween 20 (PBST) overnight at 4 °C. The next day, the cells were washed four times with PBST, each for 10 min, followed by incubation with Alexa Fluor-conjugated secondary antibody (Life Technologies), in 5% BSA/PBST for 1 h at room temperature. The cells were then washed four times in PBST, incubated with 1 µg/ml DAPI in PBS for 5 min, and washed twice with PBS. The slides were mounted with ProLong Gold (Life Technologies), and imaged with Leica TCS SP8 fluorescent confocal microscope. Quantification of % positive cells of SAHF, p-ATM, IL8, γH2AX, and p65 was done under identical microscopy settings between samples. Cells with over 5 visible spots at the expected location were considered positive. Over 200 cells from 4 randomly-selected fields were analyzed.
3
Immunofluorescence Microscopy Protocol
4
Immunofluorescence Staining Protocol
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Immunofluorescence was performed as previously described15 (link) with slight modification. In brief, cells on the coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Cells were incubated with primary antibodies in 5% BSA in PBS supplemented with 0.1% Tween 20 first at room temperature for 30 min, and then at 4 °C for overnight. The next day, cells were probed with Alexa Fluor-conjugated secondary antibody (Life Technologies), washed and mounted with ProLong Gold (Life Technologies). The slides were imaged with Leica TCS SP8 fluorescent confocal microscope. If cells were subjected to DAPI staining, cells were incubated with 1 μg/ml DAPI in PBS for 5 min, and washed with PBS before mounting.
5
Immunofluorescence Staining Protocol
6
Visualizing CD8+ T Cell Reinvigoration
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E0771 (1.5 × 103 cells/well) were plated on Geltrex-coated 8-chamber glass slides with freshly isolated murine CD8+ T cells (1.2 × 104 cells/well) in enriched E-DMEM phenol red-free media with IL-2 (80 U mL−1), anti-mouse CD3e (2 µg mL−1, ThermoFisher, Catalog: 16-0031-86) and anti-mouse CD28 (5 µL mL−1, ThermoFisher, Catalog: 16-0281-86). After 48 h, T cell reinvigoration was performed by the addition of IL-2 (1000 U mL−1) and incubation for 3 h. Probe H5 was used at 25 μM and Ac-IEPD-CHO at 50 µM with 1 h preincubation. After 1 h treatment at 37 °C with H5 , cells were washed with media and imaged under a Leica TCS SP8 fluorescent confocal microscope (software: Leica application Suite X) under a ×40 oil objective. Excitation wavelengths: 488 nm (H5 ), 561 nm (mKate). Images were analyzed with ImageJ.
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