Hpa028417

Paraffin-embedded tissues were used to perform PHF20L1 immunohistochemistry. Briefly, tissues were dewaxed overnight at 50 °C. The specimens were hydrated in xylol (3 × 5 min) and decreasing ethanol solutions (100, 95, 90, 85, 80%; 30 s each). Antigenic recovery was carried out using 0.1 N citrate buffer at 121 °C for 20 min. Slides were rinsed 3 × with 1 × phosphate-buffered saline (PBS) for 5 min each. Endogenous peroxidase was blocked by incubating the slide in hydrogen peroxide solution for 1 h at room temperature and washed again with 1 × PBS (5 × 5 min). PBS-10% milk was added for 1 h at room temperature to block nonspecific binding. Then, the primary anti-PHF20L1 antibody (Sigma-Aldrich HPA028417) (1:100 dilution, 1 × PBS-SFB 10%) was added with overnight incubation at 4 °C. The next morning, the slides were rinsed 5 × with 1 × PBS, and the secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (HRP) was placed in a 1:100 dilution and incubated for 2 h at room temperature. Corresponding washes were performed, and diaminobenzidine substrate (Invitrogen, Catalog number 002020) was added to visualize the reaction followed by incubation for 5 min. Finally, hematoxylin counterstaining was performed, and the slides were fixed for observation under a light microscope (Nikon, E100).

SKOV-3 and OVCAR-3 cells adhered to coverslips were subjected to different treatment conditions. Upon completion, the cells were fixed with 4% paraformaldehyde for 1 h at 37 °C. Cells were rinsed 3× with filtered 1× PBS and permeabilized with a 0.2% solution of Triton X-100 in 1× PBS for 15 min at room temperature with stirring. Cells were rinsed thrice with filtered 1× PBS and blocked with 10% FBS for 1 h at 37 °C. The primary antibody (Sigma-Aldrich HPA028417) was added at a 1:100 dilution in 1× PBS and incubated overnight at 4 °C in a humid chamber. On the next day, cells were rinsed 3× with 1× PBS. TRITC goat anti-rabbit (IgG) secondary antibody (ab50598, Abcam) was added to OVCAR-3 cells and incubated for 1 h at 37 °C under protection from light. For the SKOV-3 assays, a different secondary antibody was employed: Alexa Fluor 647 donkey anti-rabbit (IgG) secondary antibody (1:100 dilution) (711-605-152, Jackson InmunoResearch Laboratories), this with the objective to improve the signal of the protein. Vectashield with DAPI (Cat. No. H-1200) was added, and the coverslip was mounted on a slide. The assembled samples were stored at − 20 °C protected from light until observation under a Carl Zeiss LMS 700 confocal microscope. ZEN 2012 software was used to analyze the samples (Carl Zeiss).