Anti c rel

IHC was performed on paraffin-embedded sections according to standard protocols. Briefly, tissues were fixed in 4% paraformaldehyde solution or 10% neutral-buffered formalin at room temperature overnight and paraffin-embedded following standard procedures. The sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol (100, 90, 80, 75%) for 3 min each time and microwaved-heated in sodium citrate buffer for antigen retrieval. Then, the sections were blocked in 5% BSA and incubated with primary antibodies: anti-LDHA (1:100 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA #3582), anti-CD33 (1:100 dilution, ProteinTech Group, Inc., Wuhan, China, #17425), anti-LOX-1 (1:200 dilution, ProteinTech Group, Inc., #11837), or anti-c-Rel (1:100 dilution, Santa Cruz Biotechnology, USA, #sc-6955), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Antibody binding was visualized using a 2-Solution DAB Kit (Invitrogen). The images were obtained with an inverted microscope (Olympus IX71, Japan). An H-score was calculated using the following formula: H-score = ∑ (PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + percentage of cells of strong intensity × 3). Here H-score was recorded as a continuous variable.

ChIP assays were performed by using SimpleChip Enzymatic Chromatin IP Kits from Cell Signalling (Boston, MA, USA) due to the manufacturer's instructions. DNA from Panc1 or Patu8988t cells was submitted to immunoprecipitation with 2 μg of mouse IgG (Upstate, Darmstadt, Germany), anti-c-Rel (Santa Cruz Biotechnology), anti-Rbp1, anti-NFATc2 or rabbit IgG (all Cell Signalling). Primers for real-time PCR: NFATc2-F (5′-CGAACCAGGGTCTAGACAAG-′3); NFATc2-R (5′-CCATGCAGGTGTCTGAAGTA -′3) (from MWG Eurofins).

Cells were washed in PBS and fixed in 4% PBS–PFA for 10–15 minutes at RT. For p65 and c-Rel immunocytochemistry cells were incubated in ice cold methanol for 10 minutes at -20°C. For HMGB1 staining slides were incubated in Citrate buffer and subjected to microwave heating at 350 W for 5 minutes, then left to reach RT for 20 minutes and washed in PBS. Slides were incubated in blocking solution containing 10% donkey serum or 10% goat serum (depending on the source of the secondary antibody) in PBS for 1 hour at RT. Subsequently, cells were incubated overnight at 4°C with the following primary antibodies: anti-CD11b 1:100 (OX-42, Millipore), anti-p65 1:30 (BD Biosciences), anti-c-Rel 1:50 (sc-70, Santa Cruz), anti-HMGB1 1:100 (ab79823, Abcam). After rinsing, slides were incubated with the appropriate secondary antibodies for 1 hour at RT. Cells were washed in PBS and mounted in mounting medium Vectashield (Vector Laboratories). Negative control slides were incubated with blocking solution instead of the primary antibodies. Staining was visualized in a fluorescence microscope (Axiophot; Carl Zeiss, Jena, Germany). Quantification of fluorescence intensity was done with ImageJ Software (NIH, USA).