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Free of Acrylamide Sodium Fast Free-of-Acrylamide Clearing Tissue (FACT) is a developed technique using no acrylamide for clearing tissues. As the lipid removal normally is a harmful process and it causes loss of biological molecules such as proteins and on the other hand is crucial for transparency and efficient antibody staining throughout the whole tissue especially for microscopy and imaging, the FACT technique is suitable since it makes chemical bonding of membrane and intracellular proteins with the extracellular matrix creating a massive three-dimensional (3D) matrix and structural support to fortify the tissue during processing. Compared to other acrylamide-based techniques, FACT requires less labor, toxic, and harmful chemicals. Here we describe protocols encompassing every angle and dimension of the FACT protocol for antibody staining and imaging of whole-cleared tissues while preserving the structure and increasing the image quality. The entire protocol includes tissue perfusion, fixation, clearing, antibody staining, Refractive Index Matching (RIM), microscopy, and imaging; this timing varies due to the size, weight, different kind of tissues and the type of immunostaining. This technique has been favorably performed on different types of tissues for molecular interrogation analysis of large tissues. |