En-Bloc Tissue Staining Protocol

Large-volume en-bloc staining was performed as follows (also see Supplementary Table 1 for details). Tissue was first immersed in 2% OsO₄ aqueous solution (Serva) buffered with cacodylate (0.15 M, pH 7.4) at room temperature for 90 min. The staining buffer was then replaced by 2.5% ferrocyanide (Sigma-Aldrich) in 0.15 M cacodylate buffer (pH 7.4) and incubated at room temperature for another 90 min. Sequentially, the tissue was incubated in filtered thiocarbohydrazide (saturated aqueous solution at room temperature, Sigma-Aldrich) at 40 °C for 45 min, 2% unbuffered OsO₄ aqueous solution at room temperature for 90 min and 1% UA (Serva) aqueous solution at 4 °C overnight. Double rinses in nanopure filtered water for 30 min each were performed between the ferrocyanide and thiocarbohydrazide step, the thiocarbohydrazide and OsO₄ step, and the OsO₄ and UA step. On the next day, the tissue (still in UA solution) was warmed up to 50 °C (oven) for 120 min. After being washed twice in nanopure filtered water at room temperature for 30 min, the tissue was incubated in a lead aspartate solution at 50 °C for 120 min. The lead aspartate solution was prepared by dissolving 0.066 g lead nitrate (Sigma-Aldrich) in 10 ml 0.03 M aspartic acid (Serva) and pH adjusted to 5 with 1 N KOH. The tissue was then washed twice in nanopure filtered water for 30 min. The image in Supplementary Fig. 2b was taken from a tissue block stained with the same procedure described above except that the 120 min 50 °C incubation in UA was omitted.