Affinipure fab fragment donkey anti mouse igg h l

Adult mice were transcardially perfused with paraformaldehyde (4%) to fixate tissue and brains were extracted. Gelatin-embedded slices of 40 μm thick were blocked in 3% H₂O₂ in PBS, rinsed, and for antigen retrieval incubated in sodium citrate (10 mM) at 80°C for 20 min. The slices were again rinsed and placed in blocking solution (10% normal horse serum and 0.5% Triton X-100 in PBS) for 1h at room temperature and incubated overnight at 4°C in blocking solution with AffiniPure Fab Fragment (donkey anti mouse IgG (H+L), 1:200, Cat# 715-007-003, Jackson ImmunoResearch). The next day, slices were incubated for 48–72h with primary antibodies (anti-CAMKII alpha (6G9 clone), 1:100, NB100-1983, Novus Biologics; anti-RFP, 1:5000, 600-401-379, Rockland) shaking delicately at 4°C. Secondary antibodies (Alexa, 1:200, Jackson ImmunoResearch) were incubated for 2h at room temperature. Finally, slices were incubated with 40,6-diamidino-2-phenylindole solution (DAPI, 1:10,000, Invitrogen) for 10 min and mounted in Mowiol medium. Images were acquired with a confocal microscope (LSM700, Zeiss).

Three mAb to HRP2 (mAb 87, mAb 2G12-1C12, and mAb 1E1-A9) and an irrelevant mAb (A2C7-A3B11) were used. Previous studies showed that mouse mAb87 (IgM) detects HRP2 by metabolic immunoprecipitation and western blotting [15 (link)]; 2G12 (IgG) binds to the sequence DAHHAADAHH in the repeat region of HRP2 and 1E1 (IgM) binds to AHHAHHV [16 (link), 17 (link)]. MAb 2G12 and 1E1 work especially well together in an HRP2-based diagnostic ELISA [2 (link)]. Sequencing studies showed that epitopes recognized by 2G12 and 1E1 are present in 100% (448/488) and 99.3% (445/448) of P. falciparum isolates, respectively [17 (link)]. The mAb were used in this study to verify that these two major epitopes of HRP2 that are present in multiple strains were correctly displayed on the coupled microspheres. Serial dilutions of the anti-HRP-2 and control mAb were incubated with the HRP-2 coupled microspheres as described above. Following incubation, the microspheres were incubated with 100 μl of R Phycoerythrin-coupled AffiniPure F(ab’) Fragment Donkey Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Lab, Inc., Cat# 715-116-150) diluted in PBS + 1% BSA at 2 µg/ml.