Microwave-assisted immunostaining for rapid labeling of matrix-embedded multicellular structures

Immunofluorescence staining of cell proteins is essential to understanding biomolecular interactions within three-dimensional (3D) hydrogel cell cultures. However, the scaffold material limits passive diffusion of antibodies through thick 3D matrices, prolonging staining and washing steps and resulting in processing times that can last for several days. Microwave irradiation has previously been shown to enhance penetration of fixatives in a variety of soft tissues by increasing the rate of diffusion through the sample, yet it is unknown if microwave irradiation can improve immunofluorescence staining of cells in 3D hydrogel cultures. Here, we demonstrate a microwave-assisted immunostaining technique that rapidly labels cells within spheroid structures embedded within thick intact hydrogels. These results show that collagen-embedded breast epithelial spheroids can be efficiently labeled with primary antibodies in less than 3.5 h. We show significantly enhanced staining and greater depth penetration with microwave-assisted staining compared to conventional benchtop staining methods. We demonstrate staining of collagen-embedded breast cancer spheroids with complete staining achieved in less than 2.5 h via the microwave, which outperforms conventional staining techniques. Moreover, we demonstrate enhanced staining of spheroids embedded in basement membrane-derived Matrigel matrices with the microwave method compared to benchtop techniques. Finally, we directly compare 2-h microwave-assisted staining to conventional 15-h longform benchtop staining and show that microwave staining increases depth penetration and intensity of stains compared to the longform staining. This work develops a microwave-assisted staining protocol that provides a rapid and reproducible method to label a variety of cell types within various 3D hydrogel cell culture systems.