Confocal microscopy showing colocalization of SHP1 and phosphorylated NPM-ALK into cytoplasmic granules. (A) Karpas 299 cells (A, low-power magnification; C, high-power magnification) and tissue sections (B,D) from the ALCL tissue microarrays were stained with ALK1 and anti-SHP1 (A-B) or with ALK1 and anti-pY664 NPM-ALK (C-D) antibodies. Antibody binding was visualized with conjugated goat antimouse or goat antirabbit, respectively, as described in “Materials and methods.” ALK1 antibody (red) shows diffuse and granular (arrows) cytoplasmic (Cy) staining associated with a strong nuclear (N) and nucleolar (arrowhead) staining (Ai,Aiv,Bi,Ci,Di). Comparable cytoplasmic staining pattern is observed after SHP1 staining (Aii,Av,Bii,Cii,Dii) and the 2 signals are colocalized (merge; Aiii,Avi,Biii, Ciii,Diii). Note that SHP1 (green) is essentially detected in the cytoplasm with a weak associated nuclear staining. Anti–NPM-ALK phosphorylated on Y664 staining (green) is restricted to the cytoplasm and clearly concentrated into cytoplasmic granules (arrows; C-D). The colocalization of the 2 signals is shown (Ciii,Diii). Note that a comparable cytoplasmic staining is seen on Karpas cells (C) and on tissue sections (D) after staining with ALK1 and anti-pY664 NPM-ALK antibody. Results shown are representative of 3 independent experiments. Total magnification is 63 × for Ai-iii; 170 × for Aiv-vi and all subpanels of B, C, and D.