Figure 4.

Immunofluorescent labeling of hIAPP, hIAPP variants, and rIAPP in COS-1 cells. Cells were transfected with the pMT2-hIAPP (A and B), pMT2-hIAPP^anti (C), and co-transfected pMT2-hIAPP and pMT2-hIAPP^anti expression vectors as described in Materials and Methods and cultured in 8-well chamber slides for 48 hours. Immunofluorescent labeling on permeabilized cells was performed with rabbit anti-amylin antibodies and FITC-labeled GAR secondary antibodies. Phase contrast (B) and immunoflouresence microscopy (A, C, and D) were performed concurrently on a Zeiss laser scanning confocal microscope. Intense immunofluoresence (green) is seen localized in clusters in the perinuclear regions of hIAPP-expressing cells only (A). Cells were also transfected with the nonamyloidogenic pMT2-hIAPP^mut (E) or pMT2-rIAPP (F) expression vectors. After 48 hours, the permeabilized cells were labeled with rabbit anti-IAPP antibodies and GAR-FITC to detect intracellular IAPP (Materials and Methods) by LSCM. Apoptotic bodies (G) and membrane blebbing (H) extruding from cells undergoing apoptosis in pMT2-hIAPP-transfected COS-1 cells at 48 hours after transfection. Translocated extracellular phosphatidylserine residues were labeled by annexin-V and Rhodamine Red fluorescence and analyzed by LSCM. Annexin positive labeling on apoptotic cells (red staining) and formation of apoptotic bodies (arrows) are shown in G. Intracellular hIAPP is also evident in apoptotic blebs (green staining and arrows) as labeled with anti-amylin antibodies and FITC fluorescence in H.