Cells were plated in 35-mm glass bottomed culture dishes (Mattek), and were cultured overnight. The cells were then left untreated (DMSO) or treated with 10 μM Rucaparib or iRucaparib for 48 hrs before washing with PBS. Cells were fixed with 4% Paraformaldehyde for 15 min at RT and were washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min and were blocked with 1% BSA in PBS for 30 min. Fixed cells were incubated with a γH2AX antibody (1:200, Cell Signaling Technology, #9718) at 4℃ for overnight, washed three times with PBS for 5 min, and incubated with an Alexa Fluor 488 conjugated anti-rabbit antibody (1:500, Thermo Fisher Scientific, A-11008) for one hour at RT. Cells were washed three times with PBS for 5 min and stained with DAPI (1:1000, Thermo, #62248) for 2 min. Cells were washed with PBS and mounted with the FluorSave reagent (Millipore, #345789). Images were collected on a Zeiss LSM 880 Airyscan inverted confocal microscope.
Lsm 880 airyscan inverted confocal microscope
Manufactured by Zeiss
The LSM 880 Airyscan is an inverted confocal microscope manufactured by Zeiss. The core function of this device is to provide high-resolution imaging capabilities through the use of the Airyscan detection system.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Fluorsave reagent, by Merck Group (3 mentions)
γh2ax antibody, by Cell Signaling Technology (2 mentions)
35 mm glass bottomed culture dishes, by MatTek (2 mentions)
Alexa fluor 488 conjugated anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Sc 9996, by Santa Cruz Biotechnology (2 mentions)
Pa1 10033, by Thermo Fisher Scientific (1 mentions)
Mca2887, by Bio-Rad (1 mentions)
Ap0785, by ABclonal (1 mentions)
Mouse α tubulin, by Merck Group (1 mentions)
Zen black, by Zeiss (1 mentions)
Glass bottomed culture dishes, by Ibidi (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
A32740, by Thermo Fisher Scientific (1 mentions)
T5168, by Merck Group (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
T7451, by Merck Group (1 mentions)
Alk d5f3, by Cell Signaling Technology (1 mentions)
1 phenyl 2 thiourea ptu, by Merck Group (1 mentions)
Low melting point agarose, by Merck Group (1 mentions)
8 protocols using lsm 880 airyscan inverted confocal microscope
1
Quantification of DNA Damage Response
2
Immunofluorescence Microscopy of Cultured Cells
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Cells grown on acid‐etched glass coverslips were washed with PBS and fixed with 3.7% formaldehyde in PBS buffer for 10 min. Cells were kept in blocking buffer (3% BSA in PBS) for 1 h and incubated for 2 h or overnight with primary antibodies diluted in 3% BSA/PBS buffer followed by 1‐h incubation with secondary antibodies (Table 2 ). Primary antibodies were against, α‐tubulin mouse (1:1,000; Sigma), α‐tubulin rabbit (1:800; 18251; Abcam), GFP (1:1,000; 6556; Abcam), GFP (1:1,000; sc‐9996; Santa Cruz Biotechnology), ALK rabbit (D5F3) (1:100; CST), ALK (31F12) mouse (1:100; CST), GRB2 rabbit (1:1,000; PA1‐10033; Invitrogen), GRB2 (1:100) mouse (C7; Santa Cruz Biotechnology), SOS1 mouse (1:100; MCA2887; Bio‐Rad;), PLCγ2 (Y759) rabbit (1:100; AP0785; ABclonal), PI3K p85β mouse (1:100; 1B180967; Abcam), c‐KIT (Y721) (1:100; 44‐494G; Thermo Fisher Scientific). Secondary antibodies were Alexa Fluor‐488 and ‐594 goat anti‐rabbit and goat anti‐mouse IgGs (1:200; Invitrogen). Imaging was performed on a Zeiss LSM880 + Airyscan Inverted confocal microscope using a 40× oil objective (numerical aperture, 1.4). Z‐stacks comprising of 10–20 ×0.3 µm sections were acquired. Images were analysed using ImageJ (v.2.0.0).
3
Quantification of DNA Damage Response
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Cells were plated in 35-mm glass bottomed culture dishes (Mattek), and were cultured overnight. The cells were then left untreated (DMSO) or treated with 10 μM Rucaparib or iRucaparib for 48 hrs before washing with PBS. Cells were fixed with 4% Paraformaldehyde for 15 min at RT and were washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min and were blocked with 1% BSA in PBS for 30 min. Fixed cells were incubated with a γH2AX antibody (1:200, Cell Signaling Technology, #9718) at 4℃ for overnight, washed three times with PBS for 5 min, and incubated with an Alexa Fluor 488 conjugated anti-rabbit antibody (1:500, Thermo Fisher Scientific, A-11008) for one hour at RT. Cells were washed three times with PBS for 5 min and stained with DAPI (1:1000, Thermo, #62248) for 2 min. Cells were washed with PBS and mounted with the FluorSave reagent (Millipore, #345789). Images were collected on a Zeiss LSM 880 Airyscan inverted confocal microscope.
4
Immunofluorescence Imaging of HeLa Cells
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After the treatment with DMSO or nimbolide, HeLa cells were washed once with 1× PBS and then fixed with 4% paraformaldehyde for 20 min at RT, followed by three times wash with 1× PBS. The cells were permeabilized with 0.25% Triton X-100 in 1× PBS for 10 min and blocked with 1× PBS containing 2% bovine serum albumin for 1 hour. Fixed cells were incubated with primary antibodies at 4°C overnight, followed by the incubation with the fluorescent secondary antibody for 1 hour at RT. Cells were washed three times with 1× PBS for 5 min and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for 2 min. DAPI was used to visualize the nuclei. Cells were washed with 1× PBS and mounted with the FluorSave reagent (Millipore). The fluorescence images were then collected with a Zeiss LSM 880 Airyscan inverted confocal microscope.
5
Confocal Imaging of Brain Cryosections
6
Live-cell Imaging of Drug Dynamics
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Time‐lapse imaging was performed using a Zeiss LSM880 + Airyscan Inverted confocal microscope using a 40× oil objective and a scan zoom of 4. Cells were cultured in glass‐bottomed culture dishes (Ibidi) and maintained at 37°C in an atmosphere supplemented with 5% CO2 using a ZL multi S1 Incubator box system. Drugs were added just before imaging with pre‐warmed OptiMEM (Thermo Fisher Scientific) containing 10% FBS. Z‐stacks comprising of twenty 1 μm steps were acquired every 1 s for 1 min. Stacks were processed into maximum intensity projections and movies prepared using ImageJ (v.2.0.0).
7
Immunofluorescence Staining of Tubulin
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Cells grown on acid-etched glass coverslips were washed with PBS and fixed with 3.7% formaldehyde in PBS buffer for 10 min. Cells were kept in blocking buffer (3% BSA in PBS) for 1 h and incubated for 2 h or overnight with primary antibodies diluted in 3% BSA/PBS buffer followed by 1 h incubation with secondary antibodies. Primary antibodies were against α-tubulin mouse (1:1000, T5168; Sigma-Aldrich), α-tubulin rabbit (1:800; 15246; Abcam, Cambridge, UK), Acetyl-α-tubulin (1:2000, T7451; Sigma-Aldrich), GFP (1:1000, sc-9996; Santa Cruz-Biotechnology) and ALK (D5F3) (1:100, 3633; Cell signalling Technology) (Table S2 ). Secondary antibodies were Alexa Fluor-488 and -594 anti-rabbit and anti-mouse goat IgGs (1:200, A32723; A32740; Invitrogen). Imaging was performed on a Zeiss LSM880 + Airyscan Inverted confocal microscope using a 40× oil objective (numerical aperture, 1.4). Z-stacks comprising of 10–20 × 0.3 µm sections were acquired. Images were analysed using ImageJ (v.2.0.0).
8
Live Imaging of Zebrafish Embryos
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Fixed or live embryos were selected for fluorescence signal, anaesthetized in E3 fish water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.4) with 1x tricaine (0.08%, Sigma) and mounted in glass bottom Petri dishes (MatTek) using 0.7% low-meltingpoint agarose (Sigma) containing 1x tricaine. For live-imaging, E3 with 1x tricaine and 0.003% 1-phenyl-2-thiourea (PTU, Sigma) to avoid pigmentation, was added to the dish. A Zeiss LSM880 Airyscan inverted confocal microscope was used for live-imaging and imaging of fixed samples. For fixed samples, the 40x (NA = 1.3) silicon oil objective was used. Z-stacks were made with a step size of 0.5 µm. For live imaging, the 25x (NA = 0.8) oil objective was used. Images were acquired with a zoom of 1-1.6 and z-stacks were made with a step size of 0.5 to 1.0 µm. Frames were acquired every 5-30 minutes. High resolution timelapse images were acquired with an Olympus SpinSR spinning disc microscope using a 30x
(NA = 1.05) oil objective (Photometrics). Frames were acquired every 1-2 minutes with a zstack step size of 0.7 µm. Images were analyzed with the ImageJ software.
(NA = 1.05) oil objective (Photometrics). Frames were acquired every 1-2 minutes with a zstack step size of 0.7 µm. Images were analyzed with the ImageJ software.
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