Detection of 29-15–positive (29-15⁺) germinal center (GC) B cells.

(A) The immunohistochemical analysis was carried out on Peyer's patches with the 29-15 monoclonal antibody (mAb) and alkaline phosphatase (ALP) antirat Ig antibody (Ab). Positive cells, stained with Vector Blue ALP substrate, appear in the central area . The strong signal in the surrounding area is in intestinal villi containing nonspecific endogenous ALP activity. For 2-color staining, the sections were further stained with either biotin–anti-B220 mAb or biotin–anti-IgD mAb followed by horseradish peroxidase-streptavidin in combination with 3,3′-diaminobenzidine tetrahydrochloride. (B) The sections of the GC area from mice immunized with sheep red blood cells (SRBC) were stained with PNA, anti-bromodeoxyuridine, and the 29-15 mAb in combination with the individual colors. The upper panel shows hematoxylin staining of the GC area and the central artery (CA). The middle panel shows 3-color staining, indicating 29-15⁺PNA-positive (PNA⁺) cells. The lower panel indicates the location of 29-15⁺PNA⁺ cells. (C) The sections from Bcl6-positive (Bc6^+/+) reconstitution marrow (RM) and Bcl6-negative (Bc5^−/−) RM after immunization were doubly stained with the 29-15 mAb and PNA. No 29-15⁺PNA⁺ cells were observed in Bcl6^−/− RM. (D) Sections from SRBC-immunized mice were analyzed with the 29-15 mAb (blue) and anti-CR1 mAb (brown). Two-color staining shows the close contact of 29-15⁺ cells with CR1-positive follicular dendritic cells (FDC). The magnification in the photomicrograph at the lower left is × 125; that in the photomicrograph at the lower right is × 320.