Gcet1 validation and characterization in reactive lymphoid follicles. (A,B) Cytospin slides of transfected HEK cells. The transfected HEK cell line with GCET1-cDNA showed a specific staining with Gcet1 mAB. However, the same line with GCET2-cDNA was negative with Gcet1 mAB. (C-F) Double immunofluorescence combined with enzymatic staining in reactive follicles. Gcet1 was found to be expressed in a relatively large proportion of BCL6-positive cells, whereas few BCL6-positive cells were negative with Gcet1 (C,D). Double immunostaining for both Ki67 and Gcet1 demonstrated that all Gcet1⁺ cells were double-positive with Ki-67, whereas some Ki67-positive cells were negative with Gcet1 (E,F). (G,H) Immunostaining of a reactive follicle with anti-Gcet1 mAb. Gcet1 expression was restricted to germinal center B cells. Granular cytoplasmic staining was observed in centroblasts (large noncleaved cells) and large centrocytes (large cleaved cells) but not in small centrocytes (small cleaved cells). (I-K) The same GC is shown with Bcl6, CD10, and Gcet1 Abs for comparison. Gcet1 mAb had a heterogeneous staining pattern, selecting a subpopulation of Bcl6-CD10 double-positive cells. (L) Double immunoenzymatic staining with Gcet1 (red) and MUM-1/IRF4 (brown) demonstrated a small population of Gcet1-MUM1/IRF4 double-positive cells (red arrows). Blue and green show Gcet1-only or MUM1/IRF4-only positive cells. respectively.