Fixed specimens were dehydrated, infiltrated and embedded in eosin dyed methacrylate resin. For dehydration, the specimens were transferred into an ascending methanol series (10% increments until 90%, then 95% and 100%, 1 h each, constant gentle rocking). Following dehydration, the specimens were infiltrated with JB-4 infiltration solution (Polysciences, Inc., Warrington, PA, USA) for 72 h and embedded in JB-4 embedding solution. Eosin B (Sigma-Aldrich, St. Louis, MO, USA) (0.275 g/100 mL) and acridine orange (Sigma-Aldrich, St. Louis, MO, USA) (0.055 g/100 mL) were added to both solutions [9 (link)].
For embedding, the specimens were transferred from the infiltration solution into embedding molds filled with embedding solution. Embryos were placed, head down and carefully positioned with their cranio-caudal axis perpendicular to the future block surface as soon as the viscosity of the embedding solution started to increase. For this, forceps or a blunt needle were used. As soon as the embryos were fixed by the hardening resin, block holders were placed on the embedding molds. Then, the molds were fully filled and sealed air proof using mineral oil to prevent oxygen to affect polymerization. After polymerization at room temperature for 12–24 h, the resin blocks were baked at 90 °C for 24–48 h to speed up the hardening process [9 (link),23 (link)].
For embedding, the specimens were transferred from the infiltration solution into embedding molds filled with embedding solution. Embryos were placed, head down and carefully positioned with their cranio-caudal axis perpendicular to the future block surface as soon as the viscosity of the embedding solution started to increase. For this, forceps or a blunt needle were used. As soon as the embryos were fixed by the hardening resin, block holders were placed on the embedding molds. Then, the molds were fully filled and sealed air proof using mineral oil to prevent oxygen to affect polymerization. After polymerization at room temperature for 12–24 h, the resin blocks were baked at 90 °C for 24–48 h to speed up the hardening process [9 (link),23 (link)].
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