Each group comprised five corneal buttons. Two buttons were used for scanning electron microscopy (SEM, Hitachi, H-450), and the other three buttons were used for transmission electron microscopy (TEM, JEOL-100CXII). The main procedure of the electron microscopy was performed by professional staff at our hospital.
In the SEM procedure, the corneal buttons fixed in 4% glutaraldehyde for more than 1 h, washed in a buffered solution of 0.2% sucrose-cacodyl for 4−10 h, postfixed in 1% osmium tetroxide in veronal acetate buffer for 1 h, and dehydrated through an ethanol series. The samples were then dried and mounted on SEM stubs using carbon adhesive tabs. They were then sputter-coated with a 10-nm thick layer of gold and examined with a scanning electron microscope (Hitachi, H-450).
In the TEM procedure, the corneas were fixed in the 2% glutaraldehyde for more than 24 h at 4 °C, washed in PBS three times, postfixed in 1% osmium tetroxide for 2 h, dehydrated through an acetone series, and then embedded in Epon. Ultrathin Epon sections 50–70 nm thick were cut. The samples were contrasted with 5% uranyl acetate and lead citrate and examined with TEM (JEOL-100CXII).
In the SEM procedure, the corneal buttons fixed in 4% glutaraldehyde for more than 1 h, washed in a buffered solution of 0.2% sucrose-cacodyl for 4−10 h, postfixed in 1% osmium tetroxide in veronal acetate buffer for 1 h, and dehydrated through an ethanol series. The samples were then dried and mounted on SEM stubs using carbon adhesive tabs. They were then sputter-coated with a 10-nm thick layer of gold and examined with a scanning electron microscope (Hitachi, H-450).
In the TEM procedure, the corneas were fixed in the 2% glutaraldehyde for more than 24 h at 4 °C, washed in PBS three times, postfixed in 1% osmium tetroxide for 2 h, dehydrated through an acetone series, and then embedded in Epon. Ultrathin Epon sections 50–70 nm thick were cut. The samples were contrasted with 5% uranyl acetate and lead citrate and examined with TEM (JEOL-100CXII).