Figure 2

For immunohistochemistry, an antibody against activated human/mouse Caspase 3 (dilution 1:50) was used for the detection of apoptosis in the different tissues at different time-points. Heart: A, B: Representative stainings for heart tissue of BD prior to perfusion (A) and after 4 h of CIT (B) against activated Caspase 3. C, D: Representative stainings of nitrotyrosine positive cells in BD hearts prior to perfusion (C) and after 6 h of CIT (D). Nitrotyrosine positive cells were more common in BD hearts after CIT, at all other time points no difference between LD and BD was found. Liver: E, F: Representative stainings of liver tissue of BD prior to perfusion (E) and after 6 h of CIT (F) against activated Caspase 3. G, H: Representative stainings for nitrotyrosine positive cells of BD liver tissue prior to perfusion (G) and after 6 h of CIT (H). Kidney: I, J: Representative stainings for kidney tissue against activated Caspase 3 prior to perfusion (I) and after 15 h of CIT (J). In kidney tissue, the number of activated Caspase 3 positive cells was significantly higher in both groups after 15 h CIT compared to prior to perfusion and was higher in BD donor organs compared to LD organs after CIT. Especially tubular tissue seemed to be more susceptible to occurrence of apoptosis after CIT. K, L: Staining for nitrotyrosine positive cells in BD kidney tissue prior to perfusion (K) and after 15 h of CIT (L). Tubular cells seemed to be more affected by nitrosative stress in BD kidney tissue after CIT. 100 x magnification. The inserts show areas of interest 200 x magnification. Arrow heads: examples of activated Caspase 3 positive cells (A, B, E, F, I, J) and nitrotyrosine positive cells (C, D, G, H, K, L); CIT: Cold ischemic time.