Sds page ready gel

Nasal mucus and serum samples were resolved on 4%–15% SDS-PAGE Ready Gel (Bio-Rad) under non-reducing conditions as described previously (9 (link), 13 (link)). For western blot analysis, the proteins on the gels were transferred onto PVDF membranes (Bio-Rad). Then, the membranes were blocked with 8% skim milk and incubated with anti-trout IgT (rabbit polyclonal antibody, pAb), anti-trout IgM (mouse monoclonal antibody, mAb) or biotinylated anti-trout IgD (mouse mAb) (9 (link)) antibodies followed by incubating with peroxidase-conjugated anti-rabbit, anti-mouse IgG (Invitrogen) or streptavidin (Invitrogen). For quantitative analyses of IgT, IgM, and IgD in nasal mucus and serum, immunoreactivity bands were first visualized with an enhanced chemiluminescent reagent (Advansta) and scanned by Amersham Imager 600 Imaging System (GE Healthcare), then band densitometry was analyzed with ImageQuant TL software (GE Healthcare). Finally, the concentrations of IgT, IgM, and IgD were determined by plotting the obtained signal strength values on a standard curve generated for each blot using known amounts of purified trout IgT, IgM, or IgD.

Serum and gill mucus samples were resolved on 4–15% SDS–PAGE Ready Gel (Bio-Rad) under non-reducing and/or reducing conditions. The gels were either stained with Bio-Safe Coomassie or transferred onto Sequi-Blot PVDF membranes (Bio-Rad). For western blot analysis, the membranes were blocked with 8% skim milk and incubated with anti-trout IgT (rabbit pAb), anti-trout IgM (mouse monoclonal antibody (mAb)) or biotinylated anti-trout IgD (mouse mAb) antibodies followed by incubation with peroxidase-conjugated anti-rabbit, anti-mouse IgG (GE Healthcare) or streptavidin (Thermo Scientific Pierce). Immunoreactive bands were visualized using the HyGLO Chemiluminescent HRP Antibody Detection Reagent (Denville Scientific Products). For quantitative analyses of IgM, IgD and IgT in serum and gill mucus, western blot films were scanned and the signal strength of each band was determined by using ImageQuant TL software (GE Healthcare). Thereafter, the concentration of IgM, IgD and IgT were determined by plotting the obtained signal strength values on a standard curve generated for each blot using known amounts of purified trout IgM, IgD or IgT. Original images of the western blot analyses are shown in Supplementary Fig. 11.

To access whether infected and immune fish had generated F. columnare-specific immunoglobulins, we measured the capacity of IgT, IgM, and IgD from serum, buccal mucus or BM tissue explant supernatants to bind to F. columnare using a pull-down assay as described previously (13 (link), 19 (link)). Initially, the F. columnare suspensions (1 × 10⁸ CFU ml^–1) were preincubated with a solution of 0.5% BSA in PBS (pH 7.2) at 4°C for 2 h. Subsequently, 40 μl F. columnare were incubated with diluted fluids samples (buccal mucus, serum, or BM tissue explant supernatants) separately from infected, immune and control fish at 4°C for 4 h with continuous shaking in a 300 μl volume with PBS containing 1% BSA (pH 7.2). After incubation, the bacteria were washed three times with PBS and bound proteins were eluted with 2 × Laemmli Sample Buffer (Bio-Rad) and boiled for 5 min at 95°C. The eluted material was resolved on 4–15% SDS-PAGE Ready Gel (Bio-Rad) under non-reducing conditions, and the presence of IgT, IgM, or IgD was detected by western blotting using the anti-trout IgT, IgM, or IgD antibody as described above.

Buccal mucus and serum samples were resolved on 4–15% SDS-PAGE Ready Gel (Bio-Rad) under non-reducing conditions as described previously (13 (link), 19 (link)). For western blot analysis, the gels were transferred onto PVDF membranes (Bio-Rad). Thereafter, the membranes were blocked with 8% skim milk and incubated with anti-trout IgT (rabbit polyclone antibody, pAb), anti-trout IgM (mouse monoclonal antibody, mAb), or biotinylated anti-trout IgD (mouse mAb) antibodies followed by incubating with peroxidase-conjugated anti-rabbit, anti-mouse IgG (Invitrogen) or streptavidin (Invitrogen). Immunoreactivity was detected with an enhanced chemiluminescent reagent (Advansta) and scanned by GE Amersham Imager 600 Imaging System (GE Healthcare). The captured gel images were analyzed by ImageQuant TL software (GE Healthcare). Thereafter, the concentration of IgT, IgM, and IgD were determined by plotting the obtained signal strength values on a standard curve generated for each blot using known amounts of purified trout IgT, IgM, or IgD.

To access whether the infected fish and survivor fish generated F. columnare-specific immunoglobulins, the capacity of IgT, IgM and IgD from serum, nasal mucus or tissue of olfactory organ explants supernatants binding to F. columnare was assayed using a pull-down assay as described previously (9 (link), 13 (link)). Briefly, the F. columnare suspensions (1×108 CFU ml^–1) were pre-incubated with a solution of 0.5% BSA in PBS (pH 7.2) at 4°C for 2 h. Thereafter, F. columnare (40 μl) suspensions were incubated with diluted nasal mucus, serum or tissue of olfactory organ explants supernatants from the infected fish, survivor fish or control fish at 4°C for 4 h with continuous shaking in a 300 μl volume with PBS containing 1% BSA (pH 7.2). After incubation, the bacteria were washed three times with PBS, and bound proteins were eluted with 2×Laemmli Sample Buffer (Bio- Rad) and boiled for 5 min at 95°C. The eluted material was resolved on 4–15% SDS-PAGE Ready Gel (Bio-Rad) under non-reducing conditions, then the presence of IgT, IgM or IgD was detected by western blotting using the anti-trout IgT, IgM, or IgD antibody as described above.