Goat anti chicken igm hrp

Sections were prepared as described before 1. The following antibodies were used: ms‐anti‐desmin (D33) (Thermo Scientific), the mouse‐anti chicken antibodies Cμ‐CH1 (M1), Bu‐1a, Bu1b, TCRgamma/delta (TCR1), TCRab/Vb1 (TCR2) (all Southern Biotech), and goat‐anti‐chicken‐IgM‐HRP (Bethyl Laboratories). Antibodies were detected using the Vector ABC Kit (paraffin sections) or goat‐anti‐mouse‐HRP (Jackson ImmunoResearch Europe) for 30 min (cryosections) followed by the Vector DAB Kit (Vector). Sections were counterstained with Mayer's hematoxyline (Medite). Eukitt mounting medium (EMS) was used to mount the stained sections. Pictures were taken using a Zeiss Axioskop equipped with an AxioCam MRc5 and the AxioVision software.

To measure total plasma IgM and IgY EIA plates (Corning) were coated with 2 μg/mL goat‐anti‐chicken‐IgM (Bethyl Laboratories) or 0.8 μg/mL goat‐anti‐chicken‐IgY (Sigma Aldrich) overnight at 4°C. Plates were blocked with PBS/3% skim milk and incubated with chicken plasma. Bound IgM or IgY was detected with goat‐anti‐chicken‐IgM‐HRP (Bethyl Laboratories) or goat‐anti‐chicken‐IgY‐HRP (Sigma Aldrich) and developed with TMB substrate (Thermo Fisher Scientific). OD was measured at 450 nm. To measure antigen‐specific IgM and IgY, plates were coated with 10 μg/mL KLH or BSA. For detection of the Cμ‐CH1 domain plates were coated with 2 μg/mL ms‐anti‐chicken‐ Cμ‐CH1 (M1) (Southern Biotech). All subsequent steps were performed as for detection of total plasma IgM and IgY.

Immunoglobulins were detected using: goat‐anti‐chicken‐IgM‐HRP (Bethyl), goat‐anti‐chicken‐IgY‐HRP (Jackson ImmunoResearch Laboratories), and goat‐anti‐chicken IgA (Bethyl). Blots were developed with ECL substrate (GE Healthcare) and signal detected using the MicroChemi System (DNR Bio‐Imaging Systems).