Bcip nbt substrate kit

IFN-γ ELISPOT assay was performed as previously described (14 (link)), with some modifications. Briefly, multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (An-18, eBioscience) at 5 μg/ml in PBS. Lymphocytes from immunized mice were incubated with hCD1Tg⁺ or hCD1Tg⁻ BMDCs pre-pulsed with or without MA for 18 h at 37°C. Plates were washed using PBS/0.05% Tween 20 and developed using biotinylated anti-IFN-γ mAb (R4.6A2, eBioscience), followed by streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer's instruction. IFN-γ-producing cells were quantified using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).

M. sexta PAP3 was isolated from pharate pupal hemolymph (10 (link)) and used to activate recombinant proPAP3 (32 (link)). Freshly prepared PAP3 was incubated with the purified D. melanogaster pro-cSPH242 and pro-cSPH35. The cleavage reactions, controls, and M_r markers were separated by 10% SDS and native PAGE, followed by electrotransfer and immunoblotting using antibodies against the hexahistidine tag. To better understand the process of PAP3 cleavage and high M_r SPH complex formation, aliquots of the pro-cSPHs were incubated with different amounts of PAP3 for 1 h at 37°C. The mixtures and PAP3 control were resolved by 10% SDS and native PAGE, transferred onto nitrocellulose membranes, and detected using diluted (His)₆ antiserum as primary antibody, goat-anti-mouse IgG conjugated to alkaline phosphatase (Bio-Rad) as secondary antibody, and a BCIP-NBT substrate kit (Bio-Rad) for color development. Association states of the precursors and cleaved forms of cSPH35 and cSPH242 were determined by one-dimensional electrophoresis on a series of polyacrylamide gels under non-denaturing conditions (37 ).