Ab194586

Samples were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin and cut into 4-µm sections and used for IHC staining with antibodies. To enhance antigen exposure, the slides were treated with 1X EDTA at 98°C for 10 min for antigen retrieval. The slides were incubated with endogenous peroxidase blocking solution to inhibit endogenous peroxidase and were then incubated with the primary antibody (CD14, ab183322; Abcam, dilution 1:100; MPO, ab208670; Abcam, dilution 1:1,000; NCF2, Abs135832, Absin, dilution 1:100; SOD2, ab246860; Abcam, dilution 1:1,000; PARP1, ab194586; Abcam, dilution 1:50; MUT, ab240091; Abcam, dilution 1:200; ACADM, ab239914; Abcam, dilution 1:500; PCK1, ab248573; Abcam, dilution 1:200) at room temperature for 60 min. After rinsing with Tris-buffered saline, the slides were incubated for 45 min with biotin-conjugated secondary antibody (catalog no. SA00004-2; Proteintech Wuhan Sanying, dilution 1:600), washed, and then incubated with enzyme conjugate HRP-streptavidin. Freshly prepared DAB (Zymed; Thermo Fisher Scientific, Inc.) was used as a substrate to detect HRP. Finally, slides were counterstained with hematoxylin and mounted with aqueous mounting media. Studies on human tissue samples were conducted with approval from the Ethics Committee of the Sir Run Run Shaw Hospital, Zhejiang University.

The levels of PAR, PARP1, DNA-PKcs,
Caspase 3 (C3), Caspase 9 (C9), Cleaved C3 (CC3), Cleaved C9 (CC9),
γ-H2AX, and β-actin in NPC cells were examined using western
blotting. The following primary antibodies were used as follows: anti-PAR
(Calbiochem #AM80, 100 μL); anti-PARP1 (Abcam #ab194586, 100
μL); anti-DNA-PKcs (Abcam #ab1832, 100 μg); anti-pSer2056
DNA-PKcs (Abcam #ab18192, 100 μg); anti-C3 (CST #9662, 100 μL);
anti-C9 (CST #9508, 100 μL); anti-CC3 (CST #9661, 100 μL);
anti-CC9 (CST #7237, 20 μL); antiγ-H2AX (CST #9718, 100
μL); and anti-β-actin (Abcam #ab227387, 100 μL).
Goat anti-mouse (Proteintech #SA00001-1, 100 μL) or anti-rabbit
(Proteintech #SA100001-2, 100 μL) secondary antibodies were
used. The cells were lysed on ice for 20 min in NP-40 lysis buffer
containing 1× PMSF, phosphatase inhibitors, and protease inhibitors.
Protein lysates were separated on Bis–Tris gels and then transferred
onto PVDF membranes (Millipore, USA). After blocking with 5% milk
for 1 h, the membranes were incubated with diluted antibodies overnight
at 4 °C, and then with secondary antibodies (Abcam, Cambridge,
UK) at room temperature before the protein bands were visualized with
enhanced chemiluminescence (Beyotime Biotechnology, MA, USA). Data
analysis was conducted using ImageJ software.