An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Take a murine model of breast cancer. The mammary gland tumors comprise cancer cells, cancer-associated fibroblasts, adipocytes, immune cells, and tumor vasculature.        

Intravenously inject fluorophore-conjugated tumor-specific antibodies.

Upon reaching the tumor vasculature, antibodies bind to tumor-specific transmembrane proteins on the vessel endothelium and nearby cancer cells.

Surgically isolate the mammary gland and submerge it in a freezing medium.

Obtain cryo-sections on a slide and rinse with a buffer to remove the freezing medium.

Fix the sections to preserve tissue architecture. Apply a detergent to enhance immunostaining and a blocking solution to prevent non-specific antibody binding.

Introduce a primary antibody specific for adhesion molecules on the blood vessel endothelium, and a fluorophore-conjugated secondary antibody that binds to the primary antibody, labeling the vasculature.

Add a mounting medium on the slide, and place a coverslip.

Under a confocal microscope, assess the biodistribution of tumor-specific antibodies in and around the fluorescently labeled tumor vasculature.

Following anesthetization as described in the text protocol, prepare for the tail