Alexa fluor 555 labeled anti rabbit igg

BMDCs without or with PF (5 μM) treatment were stimulated with LPS (100 ng/ml) for 30 min. Then cells were fixed with 4% paraformaldehyde for 20 min and further permeabilized with 0.1% TritonX-100 for 5 min. After washing with PBS, cells were collected for preparing cell smears. The smears were blocked with 1% bovine serum albumin for 45 min, treated with anti-NF-κB-p65 antibody (1:400) at 4 °C overnight, followed by Alexa Fluor 555-labeled anti-rabbit IgG (1:1000; Cell Signaling Technology, 4413) incubation. DAPI was used to stain the nuclei. Slides were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

U2 OS cells were cultured on glass coverslips one day before treatment with DMF or CDDP (20 μM) for 3 h, and the medium was then replaced with fresh complete medium for 12 h or 24 h to provide the appropriate conditions for DNA repair. Sides with cells were fixed using 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking, cells were incubated with primary antibodies (1:200) against γ-H2A.X, 53BP1, RAD51, PRMT5, and TRIM21 for 1 h, followed by incubation with Alexa Fluor 555-labeled anti-rabbit IgG (4413S, Cell Signaling Technology), 4’,6-diamidino-2-phenylindole (DAPI) and phalloidin-Alexa Fluor 573 (Life Technologies) overnight. Images were then acquired using a laser scanning confocal microscope (ZEISS LSM 700, Germany).