Nbt bcip

To assess whether IDO vaccination resulted in measurable T-cell responses in the two long-term patients, we performed indirect IFN- ELISPOT as previously described. Briefly, PBMCs were stimulated once in ex vivo medium +5% HS, 120 U/L interleukin-2 and 15 umol/L IDO5 peptide prior to analysis to extend the sensitivity of the assay. After 7 days in culture, cells were counted and analyzed in IFN-y ELISPOT. Nitrocellular bottomed 96-well plates (MultiScreen MAIP N45; Millipore) were coated with IFN-y capture mAb (Mabtech) overnight. Wells were washed, blocked by X-vivo medium and the effector cells were added in duplicates at different concentrations with or without 5 umol/L of the IDO5 peptide. Plates were incubated overnight and medium was discharged and wells washed prior to addition of biotinylated secondary Ab (Mabtech). Plates were incubated at room temperature (RT) for 2 h, washed and avidin-enzyme conjugate was added to each well. Plates were incubated at RT for 1 h and the enzyme substrate NBT/BCIP (Invitrogen Life Technologies) was added to each well and incubated at RT for 5–10 min. Upon the emergence of dark purple spots, the reaction was terminated by washing with tap water. The spots were counted using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers).

For each genotype, ovaries from virgin, yeast-fed females were dissected in cold 1X PBS, the ovaries were teased apart, and stage 14 oocytes were selected over the course of 2 hr. Stage 14 oocytes were homogenized in 50 µL of cold lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 6.8), 2.5 mM EDTA, 2.5 mM EGTA, 0.1% Triton-X, and protease inhibitor cocktail (Sigma-Aldrich). Ovary lysates were cleared by centrifugation twice at 14,000 rpm for 15 min at 4°C. Lysates were assayed by Bradford and concentrations adjusted before samples were combined with 2X SDS sample buffer and boiled for 5 min, and the solubilized proteins were analyzed by Western blot using standard techniques. The primary antibody used for Western blot was rabbit anti-Top2 [21] (link) at a dilution of 1∶5000 and α-tubulin (Serotec) at a dilution of 1∶5000. Immunoreactivity was detected using an alkaline phosphatase-conjugated rabbit secondary antibody (Jackson ImmunoResearch) and the nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphatase (NBT/BCIP, Invitrogen) reagents.

Serum total nitrated proteins were measured using a nitrotyrosine sandwich ELISA kit (Eastbiopharm – China) following manufacturer's instructions. Urine was concentrated (cut off 3 KDa; Amicon Millipore, Billerica, MA) followed by dot‐blot analysis to assess total nitrated proteins. Nitrocellulose membrane was loaded with 2 μL of urine, BSA (negative control), and nitrated BSA (positive control). The membrane was incubated against polyclonal nitrotyrosine antibody (1:1000) (Millipore #06‐284) for 2 h at room temperature. Nitrated proteins were detected using goat anti‐rabbit conjugated to alkaline phosphatase and NBT/BCIP (Ref. 00‐2209 – Invitrogen, Camarillo, CA, USA). ImageQuant TL (GE) was used for quantification. All samples were normalized by protein amount quantified using Bradford methods. The final results were obtained by dividing the pixels assessed in dot‐blot by total proteins and creatinine amount.