Odyssey 9120 imager

Tissues were lysed in buffer containing 1% Nonidet P-40, 50 mM Tris 3 HCl, 0.1 mM EDTA, 150 mM NaCl, proteinase inhibitors and an OGT inhibitor, TMG. Equal amounts of protein lysates were electrophoresed on 4-20% SDS-PAGE gels and transferred to Nitrocellulose membranes. Primary antibodies were incubated at 4°C overnight. Western blotting was visualized by peroxidase conjugated secondary antibodies and ECL chemiluminescent substrate with a Bio-Rad ChemiDoc Imaging System or imaged by fluorescent IgG secondary antibodies with a LI-COR Odyssey 9120 imager.

Female NU/J mice (6–8 weeks old, The Jackson Laboratory) bearing a JIMT-1/MDA-MB-231 admixed tumor were prepared in the same manner as described in the “Treatment study in a xenograft mouse model of low-HER2 heterogeneous breast cancer” section. When the tumor size reached 200–250 mm³, mice (n = 3 per group) were injected intravenously with the AF488/Cy5 dual conjugate (DOL: 2 + 2, 3 mg kg⁻¹) or a 1 : 1 cocktail of the single-dye variants (3 mg kg⁻¹ each). After 24 h, tumors were collected and fixed as described above. Cy5-based near-infrared fluorescence images of the whole tumors were taken using an Odyssey 9120 imager (Ex: 685 nm laser, Em: 700 nm channel, LI-COR). Subsequently, the tumors were processed and tissue slides were prepared as described above. AF488- and Cy5-based fluorescence images were taken using a Nikon Eclipse TE2000E wide-field fluorescence microscope (FITC and Cy5 channels). Six areas were randomly chosen for quantification using ImageJ software.

Intracranial U87ΔEGFR-luc tumor-bearing NSG mice (6–8 weeks old, male and female) were prepared as described above and randomized into three groups (n = 3) 5 days post tumor implantation. Each Cy5.5 conjugate was administered intravenously at 3 mg/kg. After 48 h, the tumor-bearing mice were anesthetized with ketamine/xylazine. Subsequently, the mice underwent cardiac perfusion with PBS(+) containing sodium heparin (10 units/mL) and then 4% paraformaldehyde/PBS(+). This step removes conjugates circulating or bound to the vascular endothelial cells. Major organs including the brain were then harvested. Cy5.5-based near-infrared fluorescence images of the harvested organs were taken using a LI-COR Odyssey 9120 imager (Ex: 685 nm laser, intensity: L1.0 for brain, L2.0 for other organs, Em: 700 nm channel). Semi-quantification of the signals from ROIs was also performed using LI-COR Image Studio software. For tissue imaging, the brain samples were embedded in paraffin and tissue sections were prepared (thickness: 5 μm). After de-paraffinization of using toluene, mounting medium containing DAPI (VECTOR #H-1200) was applied to the tissue slides. Fluorescence Images were taken using a Nikon Eclipse TE2000E inverted microscope (Cy5 channel). Three ROIs in each sample were acquired and analyzed for semi-quantification using ImageJ software.