The supernatant was stored at ?20��C. The activity Vorinostat MK0683 assay was performed at room temperature in 384-well plates in a total assay volume of 20 ��l per well. For fluorescence quantification a Safire2 (Tecan, Maennedorf, Switzerland) instrument was used with fluorescence excitation and emission wavelengths of 485 nm and 535 nm, respectively. 10 ��l of dilutions of extracts (typical range: 1500 to 12,000) were transferred to 384 well assay plates. The reaction was started by the addition of 10 ��l of a solution of the fluorogenic trypsin substrate Benzoyl-Gly-Pro-Arg-Rh110-��Glu (#BS-8378.1, Biosynthan, Berlin, Germany). The final substrate concentration was 2 ��M. The enzymatic activity in the pancreatic extract was obtained from the linear progression curves and determined after one hour.

Signal-to-background (S/B) values within the linear range of the assay signal were selected to calculate the trypsin concentrations of the extract analyzed. In order to distinguish between apparent and specific trypsin activity, the endogenous trypsin inhibitor SPINK-1 was added to the concentrated rat extracts at 10,000 fold its IC50 concentration (=1 ��M). The remaining enzymatic activity, which does not originate from trypsin, was subtracted from the apparent trypsin activity and resulted in the specific trypsin activity. The amount of trypsin was calculated by transforming S/B levels obtained in the activity assay of extract dilutions fulfilling initial enzyme velocity criteria into trypsin concentration values via a calibration curve using recombinant rat trypsin S1-94.

7 Data Analysis Imaging Experiments Each data point was captured as a stack of TIFF images acquired at 10-nm increments and was loaded into a single data structure in the memory, forming a spectral stack with a spectrum at every pixel. Using the spectral imaging software autofluorescence, food, and activated mPEG-PL-Cy5.5 probe spectra were manually selected from the spectral image by selecting appropriate regions. Using available spectral unmixing algorithms the unmixed images of ��pure�� autofluorescence, and ��pure�� mPEG-PL-Cy5.5 were created. When appropriately generated, the autofluorescence image should be uniform in intensity regardless of the presence or absence of the dye. The clean images containing only Cy5.5 fluorescent signal were used for further analysis. 7.

1 In vivo time course Region of the pancreas and liver were determined and defined from whole body clean images acquired after sacrificing GSK-3 the animals, and used on all corresponding in vivo images in the same animal. Fluorescent intensity at various time points from the same animal were normalized by fluorescence intensity determined before the start of the caerulein insult and the change in signal intensity was then plotted as a function of time. 7.