Immunofluorescence staining

5000 HT-29 cells were seeded in black Falcon clear flat-bottom 96-well plates and cultured for 3 days. Cells were fixed with 4% paraformaldehyde for 15 min at RT and blocked with 5% BSA in PBS for 1 hr at RT. Cells were then incubated with isotype control (NextCure), biotin-labeled NC410 (10 µg/mL, NextCure) or pan-collagen antibody (ThermoFisher Scientific) diluted in PBS + 1% BSA buffer for 1 hr at RT. After thoroughly washing with PBS, the slides were incubated with anti-human IgG1-AlexaFluor 594 or streptavidin AlexaFluor 594 (ThermoFisher Scientific) diluted in PBS + 1% BSA buffer for 30 min at RT. Slides were finally washed and mounted with DAPI VectaShield hardset (Vector Lab) and allowed to settle before image acquisition on a Zeiss fluorescence microscopy (Zeiss) using Zen software and ImageJ.