Horseradish peroxidase hrp conjugated anti mouse igg

PLNs from C57BL/6 WT, GlcNAc6ST-1/-2 DKO, and FucT-IV/-VII DKO mice were mashed in ice-cold PBS containing 1% Triton X-100 and Halt Protease inhibitor cocktail (Thermo Fisher Scientific) and solubilized for 4 h with rotation at 4 °C. Lysates were centrifuged at 15,000 rpm for 10 min. The supernatants were collected and protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). The obtained lysates were stored at – 80 °C until use. Equal amount of lysate was applied to each lane of the SDS-PAGE gel, and western blotting was performed using 2 μg/mL purified SF1 and 1:1000 diluted horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GeneTex, Irvine, CA, USA) that specifically reacts with undenatured mouse IgG. After incubating the membrane with Clarity Western ECL Substrate (Bio-Rad), bands were detected using the FUSION FX7.EDGE Imaging system (Vilber Lourmat, Collègien, France).

Enzyme-linked immunosorbent assay (ELISA) was used to measure ZDIII-specific IgG and IgA in mouse sera, BALFs and VFs. ELISA plates were coated overnight with 2 μg/mL ZDIII proteins at 4 °C and blocked with 1% bovine serum albumin (BSA) at room temperature (RT) for 1 h. Serial dilutions of the serum samples were added to the ELISA plates and incubated for 1 h at RT. After washing three times with PBS containing 0.05% Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:30,000, Genetex, Irvine, CA, USA), or IgA antibodies (1:10,000, Bethyl, Montgomery, TX, USA) were added. The plates were washed with PBST, developed with TMB substrate (BioLegend, San Diego, CA, USA), and the reaction was stopped with 2N H₂SO₄. The absorbance was measured using an ELISA reader at 450 nm. The end-point titers were measured as the four-fold absorbance of the negative control. BALF total IgA was measured using an IgA Mouse Uncoated ELISA Kit (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer’s instructions.