In vivo imaging system ivis

NIR images of mice under anesthesia with 2.5% isoflurane were obtained using an IVIS® In Vivo Imaging System (PerkinElmer, MA, USA) and the fluorescent intensity from the arthritic feet was quantified using the Living Images software version 4.3 (PerkinElmer, MA, USA). An ICG excitation wavelength of 745 nm and 840 nm emission filter was used. The regions of interest were chosen using an automatic operation mode. Then signal intensities were normalized to the lower NIR signal at 24 hours using this formula: $$NIRratioonthesamescanday=\frac{NIRsignalfromarthritic(atanytimes)}{thelowerNIRsignalfromarthriticmouse(at24hours)}$$

Six – eight weeks old female, athymic nude mice purchased from Envigo Laboratories, Indianapolis, USA ((order code: 069(nu)/070(nu/+)) were injected with 5 × 10⁶ breast cancer (BC) cells ((AU565: Her2+ and HCC 70 (Triple negative)) in 100μl of 1XPBS in the mammary fat pad. Mice were palpated starting at 6 days post tumor injection. Tumor weight was calculated according to this formula: weight in grams=[length in centimeters x (width in centimeters)² ]/2.
We used the IVIS in vivo imaging system (Perkin Elmer, Waltham, MA) to monitor tumor progression TAB004 antibody was conjugated with fluorophore Indocyanine green (TAB-ICG, Dojindo Molecular Technologies, MD, USA). Prior to imaging the mice, TAB-ICG in sterile saline was administered retro-orbitally (RO) in mice and imaging was conducted 24 hours post TAB-ICG injections. Mice bearing AU565 and HCC70 tumors were imaged at 21, 49 and 54 days post tumor inoculation.
Tumor fluorescence was analyzed using the Life Science Software Suite (Perkin Elmer) and region of interest were defined at tumor location. In parallel, the presence of tumor masses was assessed by palpation and recorded weekly.

Mice were imaged for up to 34 days after injection. Bioluminescence imaging was performed using a highly sensitive, cooled CCD camera mounted in a light-tight specimen box (IVIS^® In Vivo Imaging System; PerkinElmer). Animals were anesthetized with 2% isoflurane before imaging. To prepare for imaging, the mice were injected with 10 µL/10 g of body weight of luciferin (potassium salt, Xenogen, Alameda, CA, USA) 15 min prior to imaging. This dose and route of administration have been reported to be optimal for studies conducted in rodents. Images were acquired within 15 min post-luciferin administration. [26 (link)]. To perform the imaging, the mice were positioned on a heated stage within a light-sealed camera box and were continuously exposed to 1–2% isoflurane. Bioluminescence was detected with the IVIS^® camera system for 45 s, which has been shown to yield optimal results. The low levels of light emitted from the tumors were captured, digitized, and displayed. The tumor areas were identified and quantified using Living Image^® software as total photons per second. Following whole-body imaging, a laparotomy was conducted to collect blood and visualize potentially metastatic organs. The metastatic organs were either stored at −80 °C or collected in 4% PFA for immunohistochemical analyses.