Dissected tissues were fixed in 4% paraformaldehyde for 72 hours and rinsed in phosphate-buffered saline (PBS). To prepare tissues for micro-CT imaging, the tissues were incubated in a 2% Lugol’s iodine solution (6% total iodine, “diluted Lugol’s” from Thermo Fisher Scientific, Waltham, MA) for 48 hours at room temperature and then rinsed with PBS. To remove the iodine, nerves were incubated in a solution of 2.5% STS in PBS (Thermo Fisher Scientific, Florence, KY) for 48 hours, and then rinsed in PBS.
To illustrate the removal of Lugol’s by STS, normal nerve tissues were sectioned into pieces and treated with Lugol’s and then treated with either STS or PBS. For quantitative studies of the effects of Lugol’s plus STS on tissues, a single nerve from each of three animals was sectioned into two pieces that were treated in parallel with Lugol’s and STS (treated group) or PBS alone (control group). The two nerve pieces were embedded in one paraffin block such that similar areas of the same nerve were adjacent to each other and they were then sectioned and processed identically, as outlined below.
To illustrate the removal of Lugol’s by STS, normal nerve tissues were sectioned into pieces and treated with Lugol’s and then treated with either STS or PBS. For quantitative studies of the effects of Lugol’s plus STS on tissues, a single nerve from each of three animals was sectioned into two pieces that were treated in parallel with Lugol’s and STS (treated group) or PBS alone (control group). The two nerve pieces were embedded in one paraffin block such that similar areas of the same nerve were adjacent to each other and they were then sectioned and processed identically, as outlined below.