Cells were seeded on cover slides and cultured to 90% confluence maximum. Culture medium was exchanged, and half of the samples were treated with 5 µmol/L of CC-115 DNA-PK and mTor inhibitor. After 24 h of incubation at 37 °C, cells were irradiated with 10 Gy by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). After another 4 h, slides were fixed and permeabilized with 4% formaldehyde and 0.1% Triton X-100/PBS for 15 min. Slides were then blocked with 1% BSA overnight. Staining with primary antibodies mouse anti-γH2AX (1:1500, Merck, Darmstadt, Germany) and rabbit anti-Rad51 (1:250, abcam, Cambridge, UK) was carried out overnight at 8 °C [11 (link)]. Slides were further stained with secondary antibodies AlexaFluor488 goat anti-mouse and AlexaFluor594 chicken anti-rabbit (Invitrogen, Eugene, OR, USA). DAPI was applied for DNA staining (10236276001, Sigma Aldrich, St. Louis, MO, USA). Cover slides were mounted onto glass slides using Vectashield (Vector Laboratories, Burlingame, CA, US) and images were acquired by a Zeiss Imager Z2 fluorescence microscope (Zeiss, Oberkochen, Germany). Foci were quantified using Biomas Software (Version V3.07/2012, MSAB, Erlangen, Germany).
Imager z2 fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss Imager Z2 is a fluorescence microscope designed for high-resolution imaging. It features a motorized stage and autofocus system, allowing for precise control and automated operation. The microscope is equipped with a range of illumination sources, including LED and mercury vapor lamps, enabling a variety of fluorescence applications. The Imager Z2 is capable of capturing detailed images of samples stained with fluorescent markers, making it a versatile tool for researchers and scientists.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexafluor594 chicken anti rabbit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti rad51, by Abcam (2 mentions)
Mouse anti γh2ax, by Merck Group (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Isovolt titan x ray generator, by GE Healthcare (1 mentions)
Rabbit anti ki67, by Abcam (1 mentions)
Guinea pig anti vglut1, by Synaptic Systems (1 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Guinea pig anti vgat, by Synaptic Systems (1 mentions)
5 protocols using imager z2 fluorescence microscope
1
Quantifying DNA Damage and Repair after Radiation Exposure
2
Homologous Recombination Analysis in Cells
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To analyze if cells are able to use homologous recombination (HR) in an efficient manner we seeded cells onto glass slides until a confluence of 80 % was reached [19 (link),20 ]. Cells were than treated with 5 µM DNA-PK inhibitor CC-115 (Selleckchem, Houston, TX, USA) for 24 h to block the non-homologous end-joining pathway and force the cells into HR. After incubation glass slides were irradiated with a dose of 10 Gy by an ISOVOLT Titan X-ray generator and 4 h after irradiation cells were fixed with formaldehyde (4 % v/v) and blocked with a 1 % BSA containing solution. Foci of γH2AX and RAD51 were stained with primary mouse antibody anti-γ H2AX (1:1500, Merck, Darmstadt, Germany) and rabbit anti-RAD51 (1:250, abcam, Cambridge, UK). For detection secondary antibodies AlexaFluor488 goat anti-mouse and AlexaFluor594 chicken anti-rabbit (Invitrogen, Eugene, OR, USA) were used. DAPI was applied for DNA staining (10236276001, Sigma Aldrich, St. Louis, MO, USA). Images were taken by a Zeiss Imager Z2 fluorescence microscope (Zeiss, Oberkochen, Germany) and number of foci was automatically counted by Biomas software (Version V3.07/2012, MSAB, Erlangen, Germany) in minimum of 300 cells per condition.
3
Cellular response to Au-SPIONs and Citrate-SPIONs
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Cells were cultured on slides (up to 150,000 cells) for analyzing cell proliferation and DNA damage. Forty-eight hours later, the medium was exchanged, cells were treated with 2.5 µg/mL Au-SPIONs or 40 µg/mL Citrate-SPIONs in 1.5 mL medium and irradiated with 2 Gy after three hours. After treatment, the cells were incubated for 24 h at 37 °C. Then cells were fixed and permeabilized with 4% formaldehyde and 0.1% Triton X-100/PBS for 15 min and afterwards blocked with 1% BSA for 1 h at room temperature. For staining, the primary antibody mouse anti-γH2Ax (1:2500, Merck, Darmstadt, Germany) and the secondary antibody anti-rabbit Ki-67 (1:700, Abcam, Cambridge, UK) were used. DAPI was then added to stain the nucleus. Thereafter, Vectashield (Vector Laboratories, Burlingame, CA, USA) was applied to the slide and covered with a cover slip. Images were taken with a Zeiss Imager Z2 fluorescence microscope (Zeiss, Oberkochen, Germany). Electromagnetic convergence points (foci) generated with anti-γH2Ax were quantified using Biomas software.
4
Immunocytochemical Analysis of Neuronal Markers
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Immunocytochemical analysis was performed as previously described19 (link) using as primary antibodies rabbit anti-βIII-tubulin (1:1000; Abcam, Cat#ab18207; RRID:AB_444319) and SMI31 (1:1500; BioLegend, Cat#801601; RRID: AB_2564641) or mouse anti-A2ARs (1:200; Milipore, Cat#05–717; RRID:AB_11213750) and the respective AlexaFluor-conjugated secondary antibodies (1:1000; ThermoFisher Scientific; Cat#A31572, RRID:AB_162543; Cat#A21202, RRID:AB_141607; Cat#A32728, RRID:AB_2633277). Actin cytoskeleton was labelled using Alexa Fluor-633-conjugated phalloidin (1:100; Cat#A22284). Images were acquired using Zeiss Axio Imager Z2 fluorescence microscope or LSM 710 confocal microscope with Zen Blue/Black 2012 software.
5
Immunocytochemical Detection of A2AR in Nerve Terminals
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The immunocytochemical detection of A2AR in individual glutamatergic and GABAergic nerve terminals was conducted as previously described (Canas et al., 2014 (link); Kaster et al., 2015 (link)) by double-labeling with goat anti-A2AR (1:200, Santa Cruz Biotechnology), together with either guinea-pig anti-vGluT1 (1:500, Synaptic Systems) or guinea-pig anti-vGAT (1:200, Synaptic Systems) antibodies, followed by incubation with Alexa Fluor-labeled secondary antibodies (1:500, Invitrogen). The preparations were examined under a Zeiss Imager Z2 fluorescence microscope and each coverslip was analyzed by counting three different fields and in each field an average of 500 individualized elements (Canas et al., 2014 (link)).
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