Cells were seeded into 8-chamber culture slides (BD Falcon), fixed at each time point with 4% formaldehyde at room temperature, and then rinsed with PBS and stored in PBS at 4°C Cells were permeabilized with 0.8% Triton X-100 and 0.2% BSA in PBS for 10 minutes on ice and then blocked with 1% BSA in TBS-T for 1 hour at room temperature, followed by incubation with γH2AX (1:500; Millipore) and 53BP1 (1:200; Bethyl) antibodies in 1% BSA in TBS-T for 2 hours at room temperature. The cells were then washed with TBS-T and incubated with Alexa 555 anti-rabbit and Alexa 488 anti-mouse secondary antibodies (both 1:1,000; Life Technologies) at room temperature. The cells were washed with TBS-T, rinsed in ddH20, and coverslipped with ProLong with DAPI (Life Technologies). Slides were digitally scanned with a Pannoramic Flash scanner (3DHistech) using 20×/0.8NA objective. The images were then analyzed using Metamorph software (Molecular Devices); briefly, nuclear regions were segmented using the DAPI channel, and the number of foci was counted using spot detection.
Alexa 555 anti rabbit and alexa 488 anti mouse secondary antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 555 anti-rabbit and Alexa Fluor 488 anti-mouse secondary antibodies are fluorescently labeled antibodies used to detect and visualize target proteins in immunoassays and microscopy applications. The Alexa Fluor 555 dye emits red fluorescence, while the Alexa Fluor 488 dye emits green fluorescence. These secondary antibodies are highly specific for their respective primary antibodies and provide sensitive detection of target proteins.
Automatically generated - may contain errors
2 protocols using alexa 555 anti rabbit and alexa 488 anti mouse secondary antibodies
1
Quantifying DNA Damage Foci in Cells
2
Immunofluorescence Quantification of γH2AX Foci
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The immunofluorescence staining was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). All the tissues were collected from mice as described in the immunohistochemistry section. Sections were maintained for 10 min on ice and then blocked with 1% BSA in TBS-T for 1 h at room temperature, followed by incubation with γH2AX (1:500, Millipore) antibodies in 1% BSA in TBS-T for 2 h at room temperature. The slides were then washed with TBS-T and incubated with Alexa 555 anti-rabbit and Alexa 488 anti-mouse secondary antibodies (both 1:1,000, Life Technologies) at room temperature. The slides were washed with TBS-T, rinsed in ddH20 and cover slipped with ProLong with DAPI (Life Technologies). Slides were digitally scanned with Pannoramic Flash scanner (3DHistech, Hungary) using 20 × /0.8NA objective. The images were then analysed using Metamorph software (Molecular Devices, PA); briefly, nuclear regions were segmented using the DAPI channel, and the number of foci was counted using spot detection.
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