Immunofluorescence Assay for PARP1 and XRCC1

HK-2 cells were divided into two groups: control group transfected with negative control shRNA, and another group interfered with endogenous PARP1 using shRNA vectors. The cells were placed on a 35 mm cell culture dish, PBS washed three times. They were then fixed with 4% cold paraformaldehyde for 20 min, followed by PBS washing three times. Permeabilization was performed using 0.2% Triton X-100 for 10 min, followed by PBS washing three times. Serum blocking was carried out for 30 min, followed by PBS washing three times. Cells were incubated overnight at 4 °C in the primary antibodies PARP1 (1:500, ab191217, Abcam, Cambridge, UK) and XRCC1 (1:500, ab134056, Abcam, Cambridge, UK), followed by PBS washing three times. The cells were then incubated with secondary antibodies Alexa Fluor 555 and Alexa Fluor 488 (Cell Signaling Technology, Beverly, MA, USA) for 2 h at room temperature (protected from light), or at 37 °C for 1.5 h, followed by PBS washing three times. Finally, DAPI was used to stain the nuclei, and fluorescence images were captured directly. PBS was washed off with distilled water, and the samples were mounted with glycerol and immediately imaged using an Olympus FLUOVIEW FV1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) [35 (link)].