Percoll density gradient and immunoelectron microscopic analysis of IL-1β–containing organelles.

(A) P1 and P2 pellets from PNS of DCs were pooled and subjected to a 25% Percoll density gradient. Fractions were collected from the top of the gradient and analyzed for their content in procathepsin D and mature cathepsin D (pro-CD and CD), IL-1β, or Rab7 by Western blotting and densitometry. One representative experiment of 3 is shown. Panels B and C show immunoelectron microscopic analysis of pooled P1 and P2 pellets. Sections were stained with anti–IL-1β IgG1 mAb (B) or an unrelated IgG1 mAb (C) followed by goat antimouse IgG gold–conjugated (small gold particles, arrows) and with rabbit anticathepsin D antiserum (B) or rabbit preimmune serum (C) followed by protein-A gold (large gold particles, arrowheads). Bars indicate 1 μm.