Analysis of IgM⁺ B cells during an immune response. Adult IgM1:eGFP fish were injected intraperitoneally with KLH in complete Freund’s adjuvant and then boosted 14 to 21 days later with KLH in IFA. (A) Spleens were isolated from individual fish at the indicated times and analyzed by flow cytometry for the percentage and number of GFP⁺ lymphocytes. Each point represents 1 fish, and the line represents the average response at that time point. Two independent experiments are represented in these data. The booster immunization is indicated with an arrow. (B) The gut mass was isolated from individual fish 14 days after the booster immunization and sectioned. DAPI (blue) /anti-GFP (green) immunohistochemical analyses from an unimmunized (top) and immunized (middle) fish are shown. Also shown is a compilation of the DAPI and anti-GFP stains in an immunized fish with light microscopy for context (bottom). There was an average of 10 ± 4 (SD) GFP⁺ cells in unimmunized gut sections vs 35 ± 14 (SD) GFP⁺ cells in immunized gut sections. These data are representative of results from 3 unimmunized and 4 immunized fish. The images were taken with a ×25 objective using a Leica SP5 inverted confocal microscope with LAS AF acquisition and analysis software. (C) Organs were isolated from individual adult IgM1:eGFP fish and analyzed by flow cytometry for KLH-binding cells. The percentages of KLH-binding cells from a control and immunized spleen 14 days after the booster immunization are shown. (D) Average percentages of KLH-binding cells among the IgM⁺ B cells + SD at several time points after the booster immunization (2 to 5 fish per time point). These results are representative of 2 independent experiments.