Expression of p21 and p27 in CD34⁺ blasts and in differentiating cells. (A-F) Cytospins of positively selected CD34⁺ blasts maintained overnight in low serum after harvest were stained for p21 or p27 as described (Neomarkers MoAbs). Some beads used in the immunoisolation are apparent in the isotype control. Nuclear staining of p27 is evident. Cytospins prepared after 6 days of culture in SCF, G-CSF, and IL-3 were simultaneously stained. Nuclear staining of p21 and cytoplasmic staining of p27 is evident. Cells are counterstained with Hoechst. (G-L) Staining of negatively selected CD34⁺ cells freshly harvested (G) or after 4 days of culture (H-L). (G) Confocal micrograph of cells doubly stained for CD34 (phycoerythrocin, red) and p27 (Alexa-488). A field containing a CD34⁻ cell is shown, enlarged for detail. (H) Photograph of cells doubly stained for p27 (Cy3, red) and CD71 (fluorescein isothiocyanate [FITC]). After days of culture, 86% of the cells were positive for CD71. (I) Confocal micrograph of cells doubly stained for Ki-67 (Cy3) and p27 (Alexa-48). Nuclear Ki-67 and cytoplasmic p27 is evident. Overlapping nuclear signals are yellow. (J) Isotype control for (I). All isotype controls were similarly negative. (K, L) Confocal microscopy of cells for p27, using C-terminal (G) and N-terminal (L) (Santa Cruz) antibodies.