Alexa fluor 647 donkey anti mouse igg

Neurons were fixed at DIV 10, approximately 72 h after transfection, in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature (RT). After fixing, coverslips were washed three times in PBS and incubated in blocking buffer (0.1% Triton X-100, 0.02% sodium azide, 2% normal goat serum in PBS) for 1 h at RT. Coverslips were incubated in primary antibodies [chicken anti-GFP (1:500; ThermoFisher, cat. # PA1-9533) and mouse anti-MAP2 (1:1000; BD Biosciences, cat. # 556320)] diluted in blocking buffer overnight at 4°C. After primary antibody incubation, coverslips were washed three times with PBS, after which they were incubated with secondary antibodies [Alexa Fluor 488 goat anti-chicken IgY (1:250; ThermoFisher, cat. # A-11039) and Alexa Fluor 647 donkey anti-mouse IgG (1:250; ThermoFisher, cat. # A-31571)] diluted in blocking buffer for 1 h at RT. Staining for mOrange was not necessary due to inherently high fluorescence. After secondary antibody incubation, coverslips were washed twice with PBS and incubated with Hoechst 33342 for 5 min at RT to stain nuclei. Coverslips were washed one final time with PBS and mounted onto glass microscope slides with Fluoromount G (Southern Biotechnology).

The cells were fixed with 4% paraformaldehyde in PBS, washed and permeabilized by 0.1% Triton X-100 in PBS, and non-specific binding was blocked by the Odyssey^® blocking buffer. Anti γH2AX, Ser139 (Cell Signaling Technology) was used to detect γH2AX⁺ cells. Alexa fluor 647 donkey anti-mouse IgG (Thermo Fisher Scientific) was used as secondary antibody. The nuclei were counter-stained using DAPI. Images were acquired using the Operetta CLS™ high-content system (PerkinElmer). At least 300 cells were counted. Positive foci formation of γH2AX, cells were determined by more than 3 foci per cell.

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 10% FBS in PBS for 1 h. Samples were stained with primary antibodies for overnight at 4 °C. Secondary antibody staining was performed for 1 h at room temperature with Alexa Fluor 488-donkey anti-rabbit IgG, Alexa Fluor 555-donkey anti-goat IgG, Alexa Fluor 647-donkey anti-mouse IgG, Alexa Fluor 488-donkey anti-goat IgG and Alexa Fluor 555-donkey anti-mouse IgG (ThermoFisher Scientific). Hoechst staining was performed for 2 minutes at room temperature with Hoechst solution (ThermoFisher scientific, 62249). Images were taken using Leica SP8 con-focal microscope or Olympus IX71 fluorescence microscope.

Rabbit anti-EGFR (western blot 1:1000; 4267, Cell Signaling Technology, Danvers, MA), goat anti-EGFR (western blot 1:1000; AF231, R&D Systems, Minneapolis, MN), mouse anti-pY1068 (western blot: 1:1000; immunofluorescence: 1:200; 2236, Cell Signaling Technology), mouse anti-pY845 (western blot 1:1000; 558381; BD Biosciences, Heidelberg, Germany), mouse anti-phosphotyrosine (PY72) (western blot 1:730; P172.1, InVivo Biotech Services, Henningsdorf, Germany), mouse monoclonal anti-α-Tubulin (western blot 1:4000; Sigma-Aldrich, St. Louis, MO), rabbit anti-GAPDH (western blot 1:1000; 2118, Cell Signaling Technology), living colors rabbit anti-GFP (western blot 1:1000; 632593, Clontech, Mountain View, CA), IRDye 680 donkey anti-mouse IgG (western blot 1:10,000; LI-COR Biosciences, Lincoln, NE), IRDye 800 donkey anti-rabbit IgG (western blot 1:10,000; LI-COR Biosciences), Alexa Fluor^® 647 donkey anti-mouse IgG (immunofluorescence 1:200; Thermo Fisher Scientific Inc., Waltham, MA).