Immunocytochemistry was performed using conventional methods. Briefly, cells grown on a coverslip in a 35-mm culture dish were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde/PBS for 10 min at room temperature (approximately 20°C), and then treated with 0.5% Triton X-100/PBS for 5 min on ice. After blocking the cells with 10% normal goat serum (Invitrogen, Waltham, MA; cat. 50-062Z) for 10 min at room temperature, the first antibody was applied for 30 min at 37°C. The cells were then washed, and secondary antibodies conjugated with fluorescent probes were added. The antibodies used were 53BP1 (Bethyl, Montgomery, TX; A300-272A), γH2AX (Millipore, Burlington, MA; JBW301), and pATM (CST, Danvers, MA; 4526), all of which were diluted 1000–2000×. The secondary antibodies used were TRITC goat anti-rabbit immunoglobulin G (IgG) (Jackson, West Grove, PA) and Alexa Fluor 488 goat anti-mouse or anti-rabbit IgG (Invitrogen). The pCHD7 antibody we created was also used at 1000–2000× dilution.
Alexa fluor 488 goat anti mouse or anti rabbit igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 goat anti-mouse or anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is used to detect and visualize target proteins or other biomolecules in various applications, such as immunofluorescence, flow cytometry, and Western blotting, by binding to primary antibodies raised against mouse or rabbit.
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2 protocols using alexa fluor 488 goat anti mouse or anti rabbit igg
1
Immunocytochemistry of DNA Damage Markers
2
Whole-mount Immunostaining of Zebrafish Larvae
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The whole-mount immunostaining was performed on 4-dpf zebrafish larvae with 2% PFA fixation. Antibody incubation was carried out with 4% normal goat serum and 1% DMSO in 0.3% Triton X-100/PBS for 16 h at 4°C with gentle agitation. The primary antibody was chicken anti-green fluorescent protein (1 : 750; A10263, Invitrogen). The following secondary antibodies were Alexa Fluor® 488 goat anti-mouse or anti-rabbit IgG (1 : 1000; Invitrogen).
Bright-field images were taken with a Leica DM IRB inverted microscope with a DFC 480 charge-coupled device camera and z-stacks were processed with Leica Application Suite software and Corel DRAW X3 software [34 (link)]. Immunofluorescence samples were examined using a Leica TCS SP2 AOBS confocal microscope. For excitation, an Argon laser (488 nm) was used, and emission was detected at 500–550 nm [36 (link)]. Stacks of images taken at 0.2–1.2 μm intervals were compiled, and the maximum intensity projection algorithm was used to produce final images with Leica Confocal Software and Imaris imaging software v. 6.0 (Bitplane AG, Zurich, Switzerland).
Bright-field images were taken with a Leica DM IRB inverted microscope with a DFC 480 charge-coupled device camera and z-stacks were processed with L
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