Histological observations and Biodistribution of bacteria in normal and tumor tissues
Evaluation of Bacterial biodistribution in tumor and normal tissues was carried out 48 h after tail vain injection of 108cfu/g BW25133 bacteria in mice. livers as the highest perfused normal organ and tumors were first excised and fixed in 10% formalin solution. The specimens were then paraffin embedded, sectioned and stained with gram stain and visualized at 100× using a light microscope. For counting the number of bacteria in normal and tumor tissues, liver and tumor samples were carefully weighed, homogenized in PBS and plated on LB agar. Bacterial concentration was determined by counting the number of CFUs after 24- to 48 h incubation of plates at 37 °C. Some of the sections obtained from specimens were stained with hematoxylin and eosin (H&E) staining solution to evaluate cytotoxic damages induced by cardiac peptide. Presence of BW25133 E. coli in sections was also confirmed by visualization of GFP under fluorescent microscope.
In vivo fluorescence reflectance imaging
Fluorescence reflectance imaging was utilized to initially evaluate whether bacteria could specifically localize in tumor sites and then to monitor time-course of GFP expression as the indicator of successfull expression of cardiac peptides in tumor site under hypoxic condition. Briefly, mice were IV injected with either PBS (control group) or E. coli BW25133 strain bearing construct with concentration of 108 cfu/g and images were taken on days 0, 1, 3 and 6, post injection using the FlouVison fluorescence planar imaging system in vivo  (Tajhizafarinan Noori Parseh Co., Tehran, Iran). Images from whole body were taken by placing anesthetized mice in the supine position, in the center of the imaging cassette, inside the scanning field of the system. After proper positioning of animals, the imaging cassette was set to the appropriate depth to finely confine anesthetized mice. Finally, the animal body was subjected to the laser beam and emitted non-uniform fluorescent beams were recorded using a highly sensitive thermoelectrically cooled CCD camera located on the same side of the imaged animal.