Immunofluorescent Analysis of cMet Expression

Colonoid sections were deparaffinized, and antigen retrieval was performed as described previously [37 (link)]. Tissue sections were blocked with goat serum for 30 min at RT. Overnight incubation at 4 °C was performed with the addition of monoclonal anti-cMet antibody (Abcam, #EP1454Y) at 1:100 dilution. AF488-labeled goat anti-rabbit secondary antibody (Life Technologies, Carlsbad, CA, USA; #A-11029) was applied at 1:500 dilution. The slides were stained with peptides diluted in PBS at a concentration of 5 μM for 10 min at RT after being washed with PBST. Sections were then washed with PBST and mounted with Prolong Gold reagent containing DAPI (Invitrogen, Waltham, MA, USA; #P36931). NIR fluorescence images were collected using an inverted confocal microscope (Leica, Deerfield, IL, USA; Stellaris 5). Intensities were quantified using custom MATLAB R2022a software.