Cells were treated with compounds for 3 days, following which they were adhered to glass coverslips, washed with PBS and then fixed with 3% paraformaldehyde in PBS for 20 minutes. Fixed cells were rinsed with PBS and permeabilised with 0.5% Triton-X-100 for 5 minutes. PBS washed slides were incubated for 1 hour with 10 % FCS and 0.1% Triton-X-100 in PBS following which cells were stained with an anti-53BP1 monoclonal antibody (H-300, Santa Cruz, diluted 1:600), in combination with an 8-oxoguanine antibody (2Q2311, AbCam, diluted 1:400), where indicated, in 10 % FCS and 0.1% Triton-X-100 in PBS. After rinsing with PBS coverslips were incubated with an Alexa fluor® 568 goat anti-rabbit IgG secondary antibody in combination with an Alexa fluor® 488 anti-mouse IgM secondary antibody, where indicated, for 1 hour (Invitrogen, diluted 1:400) in 10 % FCS and 0.1% Triton-X-100 in PBS. After a PBS wash, DNA was counterstained with DAPI (Sigma-Aldrich) for 10 minutes and the coverslips were mounted in Fluorescent Mounting Medium (Dako). Images were analysed with a Zeiss fluorescent microscope at 63 times magnification with supporting software.
Alexa fluor 568 goat anti rabbit igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 568 Goat Anti-Rabbit IgG Secondary Antibody is a fluorescently-labeled secondary antibody used to detect and visualize rabbit primary antibodies in immunoassays and other applications. It is conjugated to the Alexa Fluor® 568 fluorescent dye, which has an excitation/emission spectrum of 578/603 nm.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
H 300, by Santa Cruz Biotechnology (2 mentions)
8 oxoguanine antibody 2q2311, by Abcam (2 mentions)
Alexa fluor 488 anti mouse igm secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
αphospho histone h3, by Merck Group (2 mentions)
α cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Nb100 304, by Novus Biologicals (1 mentions)
P36930, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
A21429, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfap antibody, by Agilent Technologies (1 mentions)
Dabco, by Merck Group (1 mentions)
6 protocols using alexa fluor 568 goat anti rabbit igg secondary antibody
1
Immunocytochemical Analysis of DNA Damage
2
Immunocytochemical Analysis of DNA Damage
3
53BP1 Foci Formation Assay
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Cells were pre-treated with 10μM of Abiraterone, Enzalutamide or DMSO 24 hours before irradiation. Following irradiation, cells were permeabilized (0.5% of Triton X-100 in PBS) and fixed at pre-determined time points before being blocked in blocking buffer (5% FBS in PBS) and stained with 53BP1 primary antibody (1:5000) [NB100-304, Novus Biologicals (Colorado, USA)] for one hour before being washed four times and stained with Alexa Fluor 568 goat anti-rabbit IgG secondary antibody (1:2000) [A21429, Invitrogen (Massachusetts, USA)] in the dark for one hour. Following staining, cells were washed four times and mounted onto microscope slides using Prolong Gold antifade reagent with DAPI [P36930, Invitrogen (Massachusetts, USA)].
4
Immunohistochemical analysis of liver tissue
5
Histological Assessment of LFPI
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Tissue sections were processed for histological assessment of LFPI. Sham and LFPI animals were processed for cresyl violet. Slide mounting sections were briefly washed then submerged in 0.1% Cresyl Echt Violet (ScyTek Lab #CEA999). Slides were then rinsed in distilled water, followed by subsequent dehydration in graded ethanol solutions and cleared in xylene. Slides were coverslipped using permount.
Select sham and LFPI animals were processed for glial fibrillary acidic protein (GFAP) immunohistochemistry to characterize gliosis. Free-floating sections were washed in phosphate buffer and blocked in normal goat serum (NGS) for 30 min at room temperature. Following blocking, sections were incubated overnight at 4 °C in rabbit anti-GFAP antibody (1:500; DAKO ZO334) solution containing NGS and 0.3% Triton-x. Following primary antibody incubation, sections were washed then incubated in goat anti-rabbit IgG Alexa Fluor 568 secondary antibody (1:250; Invitrogen #A-11011) in the dark for 2 h at room temperature. Sections were then washed in buffer, mounted, and coverslipped with polyvinyl alcohol mounting medium with DABCO (Sigma).
Select sham and LFPI animals were processed for glial fibrillary acidic protein (GFAP) immunohistochemistry to characterize gliosis. Free-floating sections were washed in phosphate buffer and blocked in normal goat serum (NGS) for 30 min at room temperature. Following blocking, sections were incubated overnight at 4 °C in rabbit anti-GFAP antibody (1:500; DAKO ZO334) solution containing NGS and 0.3% Triton-x. Following primary antibody incubation, sections were washed then incubated in goat anti-rabbit IgG Alexa Fluor 568 secondary antibody (1:250; Invitrogen #A-11011) in the dark for 2 h at room temperature. Sections were then washed in buffer, mounted, and coverslipped with polyvinyl alcohol mounting medium with DABCO (Sigma).
6
Immunohistochemical analysis of liver tissue
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Slices of adult livers were collected and fixed in 4% PFA overnight. After dehydration, the tissues were embedded in paraffin and sectioned at 6μm. The sections were deparaffinized with xylene, rehydrated and antigens were retrieved by boiling in citrate buffer. The sections were blocked in 10% FBS and incubated with primary antibodies (αphospho-Histone H3, 06-570 Millipore; αcleaved-caspase-3, #9661 Cell Signaling) at 4°C overnight. Primary antibodies were detected with goat anti-rabbit IgG Alexa Fluor® 568 secondary antibody (A-11011, Invitrogen) and counter stained with DAPI. Images were acquired using confocal microscopy.
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