Alexa fluor 594 donkey anti rabbit igg

Cells were seeded onto 12-mm glass slides in a 24-well plates, and Olaparib treatment was given at 70% confluence. The glass slides with cells were collected after being treated for 48 h, followed by washed with PBS, fixed with 4% polyoxymethylene for 30 min, and permeabilized with 0.3% Triton X-100 for 15 min. Next, the glass slides were washed again with PBS, blocked with 5% FBS for 30 min at room temperature and incubated with the primary antibody at 4 ℃ overnight, including MRE11 (CAT#4895, CST), RAD50 (CAT#GTX70228, GeneTex), γH2AX (CAT#201082-2A9, ZENBIO), DNA-PKcs (CAT#A20837, ABclonal), DNA Ligase IV (CAT#A11432, ABclonal), KU70 (CAT#A11223, ABclonal). The secondary antibody of Alexa Fluor 488 donkey anti-mouse IgG (CAT#ANT023s, Antgene, 1:200), Alexa Fluor 488 donkey anti-rabbit IgG (CAT#ANT024s, Antgene, 1:200), Alexa Fluor 594 donkey anti-rabbit IgG (CAT#ANT030s, Antgene, 1:200) and Alexa Fluor 647 donkey anti-mouse IgG (CAT#A-21,236, Thermo, 1:1000) were performed for 1 h at room temperature. The glass slides were sealed using mounting medium containing DAPI. Cells were observed and photographed using the following equipment: fluorescence, NIKON Eclipse Ti; software: Eclipse C2, Nikon. Image-pro Plus 6.0 was used for image analysis.

Paraffin sections of formalin‐fixed samples were cut into 5 um thick sections and analyzed using an immunohistochemistry kit (Neobioscience, Wuhan, China). All sections were first incubated with IL-15 primary antibody at a 1:100 dilution (ab7213, Abcam, Cambridge, UK) at 4 °C overnight. The sections were then incubated with reagent B from the kit and finally, sections were stained with DAB working reagent for 2 to10 min depending on the degree of stain required and the cell nucleus stained with hematoxylin.
For immunofluorescence, all sections were first incubated with IL-15 primary antibody (ab7213, Abcam, Cambridge, UK) in combination with CK7 (YM3054, Immunoway, Beijing, China) or Vimentin (ab92547, Abcam, Cambridge, UK) or CD31 (1:150, ab134168, Abcam, UK) at 4 °C overnight. Then, the sections were incubated with a secondary antibody, Alexa Fluor 594 Donkey Anti rabbit IgG or Alexa Fluor 488 Donkey Anti mouse IgG (Antgene, Wuhan, China). The DNA dye-4′, 6-diamidino-2-phenylindole (DAPI), was used for nucleus staining. In the control experiments, primary antibodies were omitted. Images were captured using a fluorescence microscope (Olympus BX53, Tokyo, Japan) and the mean values of area-integrated optical densities analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Denver, USA).