Figure 3.

TNF-α induction of IL-12 p40 without a change in constitutive p35 expression in airway epithelial cells. In A, wild-type (WT) and same-strain IFN-γ (−/−) C57BL/6J mice were treated with PBS vehicle or TNF-α as described in the legend to Fig. 1. At 12 h after treatment, tracheal (rows 1 and 3) or bronchial (rows 2 and 4) tissue was fixed in formalin, blocked with nonimmune goat or rabbit serum, and then incubated with anti–IL-12 p40 or p35 Ab. For p35 immunostaining, tissues were also subjected to antigen retrieval (using 10 mM Citra solution for 10 min at 98°C). Primary Ab binding was detected by incubation with biotinylated rabbit anti–goat or goat anti–rabbit IgG, streptavidin-conjugated alkaline phosphatase complex, and a red chromogenic substrate, and tissues were counterstained with hematoxylin. Control goat nonimmune IgG gave no signal above background (data not shown). In B, wild-type C57BL/6J mice were treated with PBS as described in the legend Fig. 1. Tracheal tissue sections were incubated with control rabbit nonimmune IgG, anti–IL-12 p35 pAb, or anti–IL-12 p35 pAb in the presence of recombinant murine IL-12 p40 or IL-12 followed by detection of primary Ab binding and hematoxylin counterstaining as described in A. Bars, 20 μm.