Specimens with adhesive and mixed failures were selected for SEM analysis. The specimens were placed in aluminium stubs, sputter-coated with gold/palladium and examined at 2,000x and 5,000x with a SEM (JSM-5310, JEOL Ltd., Akishima, Tokyo, Japan) operating at 20 kV.
Specimens designated for TEM were prepared as previously described [37 (link)]. Each treated tooth specimen was restored with a flowable composite (Clearfil Protect Liner F, Kuraray Medical Inc., Tokyo, Japan), from which a 0.9 mm thick central slice containing the resin-dentin interface was retrieved by sectioning. The slices were individually demineralised in 0.1 mol/L ethylenediaminetetraacetic acid, followed by application of Karnovsky's fixative and post-fixed in 1% osmium tetroxide. Then, they were dehydrated in ethanol in ascending concentration from 50% up to 100%, immersed in propylene oxide and embedded in epoxy resin as previously described [37 (link)]. Finally, sections with ninety nanometre thickness were cut with help of an ultramicrotome, stained with 2% aqueous uranyl acetate and 1% Reynold's lead citrate, and examined with a Transmission Electron Microscope (JEM-1230, JEOL).
Specimens designated for TEM were prepared as previously described [37 (link)]. Each treated tooth specimen was restored with a flowable composite (Clearfil Protect Liner F, Kuraray Medical Inc., Tokyo, Japan), from which a 0.9 mm thick central slice containing the resin-dentin interface was retrieved by sectioning. The slices were individually demineralised in 0.1 mol/L ethylenediaminetetraacetic acid, followed by application of Karnovsky's fixative and post-fixed in 1% osmium tetroxide. Then, they were dehydrated in ethanol in ascending concentration from 50% up to 100%, immersed in propylene oxide and embedded in epoxy resin as previously described [37 (link)]. Finally, sections with ninety nanometre thickness were cut with help of an ultramicrotome, stained with 2% aqueous uranyl acetate and 1% Reynold's lead citrate, and examined with a Transmission Electron Microscope (JEM-1230, JEOL).