Cells were seeded onto coverslips in 12-well plates at a density of 150,000 cells per well the day before analysis. Following 10 Gy of IR and 6 hr recovery, cells were washed with PBS and fixed with 3% paraformaldehyde/PBS for 6 min at room temperature (RT). For staining, cells were permeabilized with 0.5% triton X-100 for 5 min on ice and then incubated sequentially with primary and secondary antibodies for 30 min each at 37°C, with 3 PBS washes in between. The primary antibodies used were PALB2 (M11), RAD51 (H-92) and BRCA1 (D9, Santa Cruz), and the secondary antibodies were FITC-conjugated goat anti-rabbit and Rhodamine-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch). Coverslips were mounted onto glass slides with VECTASHIELD Mounting Medium with DAPI (Vector Labs). Images were captured using a Nikon Eclipse TE-2000-U microscope. Images of the same group were captured with identical exposure time using NIS-Elements Basic Research software.
Rhodamine conjugated goat anti mouse antibodies
Manufactured by Jackson ImmunoResearch
Rhodamine-conjugated goat anti-mouse antibodies are secondary antibodies that bind to mouse primary antibodies. The rhodamine fluorescent dye is covalently attached to the goat antibodies, allowing detection and visualization of mouse target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te2000 u microscope, by Nikon (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Rad51 h 92, by Santa Cruz Biotechnology (2 mentions)
Brca1 d9, by Santa Cruz Biotechnology (2 mentions)
Fitc conjugated goat anti rabbit, by Jackson ImmunoResearch (2 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Mab1976z, by Merck Group (1 mentions)
3 protocols using rhodamine conjugated goat anti mouse antibodies
1
Irradiated Cell Immunofluorescence Staining
2
Quantifying αvβ3 Integrin Internalization
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αvβ3 integrin internalization was assayed using established protocols62 (link). Briefly, 24 h after plating onto glass coverslips, A549 cells were treated for 30 min on ice with antibodies against αvβ3 integrin (1:100 in culture medium, MAB1976Z, Millipore) and then again with fluorescein-conjugated goat anti-mouse antibodies (1:200 in culture medium, 115-095-166, Jackson ImmunoResearch Laboratories). This was followed by a 30 min incubation at 37 °C in culture medium supplemented with DMSO or 0.5 µM 25HC. Finally, cells were incubated for 30 min on ice with rhodamine-conjugated goat anti-mouse antibodies (1:200 in culture medium, 115-025-166, Jackson ImmunoResearch Laboratories). Samples were imaged using a TCS SP5 confocal microscope (Leica Microsystems). Internalization was quantified by calculating Spearman’s correlation between the two fluorescent channels for all pixels exceeding the background fluorescence level in the fluorescein channel.
3
Irradiated Cell Immunofluorescence Staining
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