Total nf kb

Paraffin-embedded slides were deparaffinized, dehydrated, and rehydrated using a series of acetone and alcohol washes. Antigen retrieval was performed using citrate buffer at pH 6.0 in a pressure cooker. The primary antibody (PTGS2: 160112, 1:100; Cayman Chemicals) was prepared in 4% fish gelatin and incubated with the slides overnight at 4°C. Slides were then washed and incubated with HRP-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA) at room temperature for 1 hour. DAB substrate (Invitrogen, Grand Island, NY) was used to visualize staining of protein and hematoxylin (Invitrogen) was used to stain nuclei. For immunocytochemistry experiments, Skov3-ip1 and HeyA8 cells were grown in chamber slides prior to treatment with NE. Cells were fixed in 4% paraformaldehyde and permeabilized using Triton X-100. Staining using primary (PTGES: 1:100; T6079, Sigma Aldrich, p-Nf-kB: 1:100; 3033S, Cell Signaling, total Nf-kB: 1:100, 8242S, Cell Signaling) and secondary antibodies (anti-rabbit Alexa Flour 594, 1:800; Jackson Immuno Research) was done as described above. Hoechst (Invitrogen) was used to stain nuclei.