These phenotyping studies were done with alternate draining lymph none serial sections obtained from Ag challanged mice. Tissues were incubated with 10% normal mouse serum to block nonspecific reactivity. Alternate sections were incubated with the monoclonal antibodies shown in the table. Sections were incubated at 4 degree C, washed and incubated with biotinylated mouse anti-rat F(ab')2 fragment of IgG overnight, followed by Streptavidin (SA)-FITC or as in the plate below with SA-alkaline phosphatase. The localization of Ag was accomplished on alternate sections. Controls included Ab of inapropriate specificity but the same isotype as the primary Ab used. All controls were negative. The fluorescence was evaluated in a semiquantitative manner on slides labeled with FITC conjugated SA.
NOTE that with the reagents used, ATCs and FDCs have an identical phenotype. Specifically note the cross-reactivity with MOMA-2, MIDC-8, and NLDC-145.
The reactivity of ATCs, FDC, and DCs (IDCs) is illustrated in the panel below:
The plate illustrates sections of Ag challanged mouse lymph node ATCs and IDCs labeled with the Abs shown below each micrograph. In Fig. A., the reactivity of ATCs shows the Ag transport cells forming a path toward the future site of the follicle. Based on electron microscopyc observations the lower parts of the pathway outlined by the red arrows most likely represent reactivity with FDCs. Both ATCs and FDCs are +ive for CR1 receptors and this is also shown here in Fig. A.
In Fig. B., the Ag transport site in the outer cortex is shown by the red arrows in this NLDC-145 labeled section. The lower part of the path appears to take on an oval shape typical of the developing FDC network. This structure is separated from the paracortex by an unlabeled "layer". Below this layer is the paracortex with it's many interdigitating cells, IDCs (double arrows), also heavily labeled with NLDC-145 and SA-alk. phosphatase. Thus, this section clearly demonstrates the cross reactivity between ATCs, FDCs, and IDCs.
Fig. C., shows a similar cross-reactivity between ATCs and IDCs using the monoclonal Ab MIDC-8. Note that both NLDC-145 and MIDC-8 is commonly used for the identification of DCs (specifically LDCs, Macrophages, and veiled cells (VC)). It should also be noted that NLDC-145 or MIDC-8 do not differentiate between FDCs and DCs.