Immunocytochemistry of Human Umbilical Vein Endothelial Cell (HUVEC) Spheroids in a 3D Collagen Matrix for Investigating Angiogenesis In Vitro

To begin, fix the sprouted spheroid HUVEC gels in one milliliter of 4%paraformaldehyde for one hour. Wash the gels gently in one milliliter of PBS about three to four times. Detach the gels fully from the surface of each well of the plate, then transfer them into new 24 well plates.

Add one milliliter of blocking solution to the well plate, then incubate on an orbital shaker for one hour at room temperature. After incubation, pipette 500 microliters of the primary antibody solution into the wells before incubation. The next day, wash the gels gently with PBS for five wash cycles of five minutes each.

Add 500 microliters of the corresponding secondary antibody diluted with phalloidin-FITC in blocking buffer. Incubate the plate overnight at four degrees celsius on an orbital shaker. After washing the gels in PBS, transfer them onto microscope slides.

Add two drops of DAPI-containing mounting medium onto a cover slip and place it over each gel. Seal the dried gels with nail polish. Place them in the dark before storing at four degrees Celsius until further analysis.