Validation of hydrogel expansion factors in 4 × and 12 × 3D-ExM methods. (A) Schematic diagram of the protocol of 3D-ExM. Detailed procedure is in the Materials and methods section. (B) Representative images of hydrogels before and after 4× or 12× expansion. The top view images show the diameter of the hydrogel. The side-view images show the thickness of the hydrogel. (C) Quantification of the diameter and thickness of 4× and 12× hydrogels at different time points after immersion in water. The yellow region indicates the standard deviation. n = 18 and 9 independent experiments for 4× and 12× 3D-ExM, respectively. The purple region indicates the time used for imaging in this study. 12× 3D-ExM gels reproducibly achieved 11- to 12-fold expansion in 3D (pink region).