Prolong gold reagent containing dapi

Hep3B cells were grown to ~100% confluence on cover slips. The cells were washed with PBS 3X and fixed with cold methanol for 5 min. Unlabeled peptides at concentrations of 0, 50, 100, 150 and 200 μM were added to the cells and incubated for 30 min at RT. The cells were then washed with PBS 3X, and further incubated with 5 µM of IRDye800-labeled peptides for another 30 min at 4°C. The cells were washed again with PBS, and mounted with ProLong Gold reagent containing DAPI (Invitrogen).

Cells were cultured to ~70% confluence on glass coverslips, washed with PBS, and incubated with 2% BSA in PBS for 30 min to block non-specific binding. 1 μM of Cy5.5-labeled peptides were added and incubated for 30 min at 4°C. The cells were then washed 3X with PBS, fixed with ice cold 4% paraformaldehyde (PFA) for 10 min, washed with PBS 1X, and then mounted on glass slides with ProLong Gold reagent containing DAPI (Invitrogen). For antibody staining, cells were incubated with either anti-EGFR (1:500, #2232S) or anti-ErbB2 (1:500, #29D8) primary antibody from Cell Signaling Inc overnight at 4°C after fixation, and then washed with PBS 3X and processed with secondary antibody staining. Either goat anti-rabbit IgG (H+L) labeled with Alexa-Fluor 488 (1:1000, #A-11008, Invitrogen) or goat anti-mouse IgG (H&L) labeled with Alexa-Fluor 568 (1:1000, #ab175473, Abcam) was added and incubated for 1 hour at room temperature (RT). The cells were washed with PBS 3X, and mounted onto glass coverslips. Confocal fluorescence images were collected using DAPI, AF488, AF568 and Cy5.5 filter sets. Fluorescence intensities from 3 independent images were quantified using custom Matlab (Mathworks) software.

Specific binding of KSP*-Cy5.5 to HT29 cells was validated on competitive inhibition with unlabeled KSP* peptide. Approximately 7500 HT29 cells were grown to ~70% confluence on coverslips in triplicate. Unlabeled KSP* peptide at 0, 10, 25, 50, and 100 μM was added and incubated with the cells for 30 min at 4 °C. The cells were washed 3× with PBS, and further incubated with 2 μM of KSP*-Cy5.5 for another 30 min at 4 °C. The cells were washed 3× with PBS and fixed with 4% PFA for 10 min. The cells were washed with PBS and mounted with ProLong Gold reagent containing DAPI (Invitrogen). Confocal fluorescence images were collected at each concentration, and intensities from 3 independent images were quantified using custom Matlab (Mathworks) software.

Formalin-fixed, paraffin-embedded (FFPE) human specimens of sessile serrated adenomas, adenomas, hyperplastic polyps, and normal colonic mucosa were obtained from the archived tissue bank in the Department of Pathology at the University of Michigan. 5 µm thick sections were cut, and mounted onto glass slides (Superfrost Plus, Fischer Scientific). The tissues were deparaffinized, and antigen retrieval was performed, following the standard protocol. The sections were blocked with protein serum for 15 min at RT followed by rinsing with PBS. The sections were then stained with 5 µM of KCC*-FITC for 10 min at RT. The sections were then washed for 3 min 3X with PBST and mounted with Prolong Gold reagent containing DAPI (Invitrogen) using #1 cover glass (1.5 µm thickness). The images were collected with a confocal microscope (Leica TCS SP5, Leica Microsystems) using the same exposure time for all specimens. We measured the mean fluorescence intensities from each image by placing 3 boxes with dimensions of 20×20 µm² completely within colonic epithelium using custom Matlab software (Mathworks, Inc). Regions of saturated intensities were avoided. The mean intensities were fit with a two-sample t-test. Adjacent sections were processed for routine histology (H&E) and evaluated with standard criteria by expert gastrointestinal pathologists (HDA, SO).^{9 (link)}