Granules of perforin and granzyme A are located near the cell membrane of the imDCs and are polarized toward the alloreactive CD8⁺ T-cell contact area. Representative imDC staining for perforin (green), granzyme A (red), and Hoechst 33342 (blue). Conventional triple-fluorescence staining of imDCs (A) was compared with optical sections at axial depth (z = 3μm) taken using the Zeiss ApoTome grid system (B). Typical EM appearance of imDCs (C) exhibiting round morphology with round nuclei (N) and a large number of granules (Gr). (D) Enlarged image of panel C showing evenly distributed granules in the vicinity of the cell membrane. (E) Enlarged image of the white box showing imDC inner cell morphology containing Golgi (Gol) and mitochondria (Mit). (F) imDC granules are in proximity to the cell membrane close to the microvilli (Micro) surrounding the entire cell. EM gold immunostaining for perforin inside of the granules in wild-type (i) and PKO (ii) mice (black dots marked by arrowheads). Confocal microscopy of CB6/F1 (G) or PKO (H) imDCs stained with LysoTracker Red (red) for granules, incubated with calcein AM green–stained 2C CD8⁺ T cells (green). (I) Quantification of CB6/F1 imDCs (a) versus PKO cell (b) polarization. Conjugates were scored as polarized when vesicles clearly accumulated in the area of contact. At least 50 conjugates were evaluated in each sample. (J) Interaction between specific CB6/F1 imDCs stained with LysoTracker Orange for granules (orange) and calcein AM green 2C CD8⁺ T cells (green). The elapsed time (h:min:sec) is shown at the bottom of each recorded frame. (K) Granules in imDCs containing perforin (green) and granzyme A (red) move to the area of contact. (L) TEM of conjugates between CB6/F1 imDC and 2C CD8⁺ T cells also revealing polarization of granules (Gr) toward the contact area in the nucleus (N).