Donkey anti mouse igg h l

Two kinds of anti-mouse antibodies, donkey anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA) and rabbit anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA), were used in the experiments. These antibodies were modified with biotin using the biotin labeling kit (Dojindo Laboratories, Kumamoto, Japan). The rabbit anti-mouse IgG-coated MB suspension with a concentration of ~10⁵ particles/μl was prepared by mixing 200 μl of biotinylated rabbit anti-mouse IgG at a concentration of 500 fg/μl and 200 μl of streptavidin-conjugated 25 nmϕ MBs (MP25-AV, Nanocs Inc., NY, USA) at a concentration of ~10⁵ particles/μl. The donkey anti-mouse IgG-coated PSB suspension at a concentration of ~10⁵ particles/μl was prepared by mixing 200 μl of the biotinylated donkey anti-mouse IgG with a concentration of 500 fg/μl and 200 μl streptavidin-conjugated 500 nmϕ PSBs (PS500-AV, Nanocs Inc., NY, USA) at a concentration of ~10⁵ particles/μl. The antibodies and the beads were incubated overnight at 4 °C to permit sufficient reaction time. The detection reagent was prepared by mixing 1 μl of the MB suspension, 1 μl of the PSB suspension, and 998 μl of Milli-Q water.

Mouse cortex samples were lysed in RIPA buffer containing protease inhibitors (Roche, cOmplete Mini EDTA-free Protease Inhibitor Cocktail). Cleared lysates were transferred to new tubes, and protein concentrations were determined using the Bio-Rad DC Protein Assay Kit II. For experiments analyzing secreted progranulin, conditioned media was collected from transfected HeLa cells and cleared at 10,000 x g for 10 min at 4° C. Sample buffer was added to the lysates or conditioned media, and the samples were heated at 95°C for 10 min. Equal amounts of protein lysates (100 μg) or equal volumes of conditioned media (30 μl) were separated on SDS–PAGE gels. Proteins were transferred to nitrocellulose membranes using the Bio-Rad Turbo-Blot transfer system. After blocking and antibody incubations, membranes were incubated with SuperSignal West or SuperSignal Femto chemiluminescent HRP substrate (ThermoFisher) and visualized using a Chemi-Doc system (Bio-Rad). The primary antibodies used for immunoblot analysis include: an anti-mouse progranulin polyclonal antibody (R&D Systems, AF2557, 1:200 dilution) and an anti-α-tubulin monoclonal antibody (Sigma, T9026, 1:2000 dilution). The HRP-conjugated secondary antibodies used were donkey anti-sheep IgG (H+L) (Jackson Immuno Research Labs, 713035147) and donkey anti-mouse IgG (H+L) (Jackson Immuno Research Labs, 715035150).