Figure 6.

PACT-deCAL and optimized RIMS formulation for imaging decalcified bone samples. (a) After perfusion fixation with 4% PFA, the right and left tibia bones were dissected and postfixed in 4% PFA overnight. One tibia was reserved as the uncleared control (top left), whereas the other tibia bone was A4P0-embedded and cleared (top right) according to PACT-deCAL, as follows. The tibia bone was first cleared in 8% SDS-PBS (pH 8) for 24 h, and then it was transferred into 0.1 M EDTA for 2 d and finally cleared further in 8% SDS-PBS (pH 8) for 2 d at 37 °C. The cleared bone was washed in 1× PBS three times over 1 d and incubated in PBS containing 1:200 DRAQ5 for 2 d at 37 °C. The stained bone was quickly rinsed in 1× PBS and incubated in 1.49 RIMS overnight at 37 °C. The bright-field image (top right) depicts the resulting bone transparency via the placement of a ruler (small red box) underneath the tibia, wherein the tibia's outline on top of the ruler can be seen in the magnified inset of the ruler (large red box). The cleared tibia was imaged in two regions (yellow and blue boxes) on a Zeiss LSM 780 confocal microscope with the LD LCI Plan-Apochromat 25× 0.8 NA Imm Corr DIC M27 multi-immersion objective (w.d. 0.57 mm). (b) RIMS may be formulated with different concentrations of Histodenz in order to achieve an RI that aligns with the tissue density and light-scattering properties of the sample to be imaged, as well as to the optical properties of the imaging setup (objective lens with or without immersion medium). RIMS with an RI ~1.47 is well suited for most cleared soft tissues (blue tick mark), whereas cleared bones should be incubated in RIMS with RI ~1.48–1.49 (green tick mark). Rodent husbandry and euthanasia conformed to all relevant governmental and institutional regulations; animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.