Schilling Kevin J, Huynh Katherine T, Speese Sean, Schutt Carolyn E
Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon 97201, USA.
APL Bioeng. 2025 Jul 14;9(3):036104. doi: 10.1063/5.0230800. eCollection 2025 Sep.
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.
细胞蛋白质的免疫荧光染色对于理解三维（3D）水凝胶细胞培养中的生物分子相互作用至关重要。然而，支架材料限制了抗体在厚厚的3D基质中的被动扩散，延长了染色和洗涤步骤，导致处理时间可能长达数天。先前已证明微波辐射可通过提高在样品中的扩散速率来增强固定剂在各种软组织中的渗透，但尚不清楚微波辐射是否能改善3D水凝胶培养中细胞的免疫荧光染色。在此，我们展示了一种微波辅助免疫染色技术，该技术可快速标记嵌入厚完整水凝胶中的球体结构内的细胞。这些结果表明，嵌入胶原蛋白的乳腺上皮球体可以在不到3.5小时内用一抗有效标记。与传统的台式染色方法相比，我们发现微波辅助染色的染色效果显著增强，深度渗透更大。我们证明，通过微波在不到2.5小时内即可实现对嵌入胶原蛋白的乳腺癌球体的完全染色，这优于传统染色技术。此外，与台式技术相比，我们用微波方法证明了嵌入基底膜衍生的基质胶基质中的球体染色增强。最后，我们将2小时的微波辅助染色与传统的15小时长时台式染色直接进行比较，结果表明与长时染色相比，微波染色增加了染色的深度渗透和强度。这项工作开发了一种微波辅助染色方案，该方案提供了一种快速且可重复的方法来标记各种3D水凝胶细胞培养系统中的多种细胞类型。
