Figure 4.

Flow cytometric and immunohistochemical analyses of cell types present in the inflamed joints of IIJ mice. (A) and (B) Cells were isolated from the inflamed paws of AR mice and the corresponding noninflamed paws of NAR littermates. Cells were stained with antibody mixtures and analyzed by five-color flow cytometry (n = 5 to 6 mice/group). (A) Average number of cells isolated. Error bars represent standard error of the mean (SEM). (B) Average number of cells of specific immune lineages (CD3⁺ = T cell, B220⁺ = B cell, CD11b⁺Gr-1⁺ = neutrophil, CD11b⁺Gr-1^- = macrophage). Error bars represent SEM. After gating on live, CD45⁺ cells, the percentages of each cell type were determined. This value was then multiplied by the total cells isolated from that animal to determine the final number of each cell type. (C) Immunohistochemical staining of serial sections of hindpaws from AR and NAR mice. Frozen sections were stained with biotinylated primary or isotype control antibody and detected using the Tyramide Signal Amplification Kit from PerkinElmer (staining shown in pink). The rat immunoglobulin 2a (Ig2a) isotype is the control for the anti-CD4 stain. The rat Ig2b isotype is the control for the anti-Gr-1 and anti-F4/80 stains. The mounting media contained 4',6-diamidino-2-phenylindole (staining shown in blue) for nuclear stain. AR mice were between 80 and 95 days old.