2.5.3. Microstructural Analysis

Control and PEF-treated kernels were fixed employing the procedure reported by Kendall et al. [21] with modifications. Glutaraldehyde and osmium tetroxide solutions were made using 0.1 M phosphate buffer (pH 7.2). Kernels were fixed in 2.5% glutaraldehyde solution and left overnight. Next, samples were washed three times in 0.1 M phosphate buffer and postfixed in 1.0% osmium tetroxide solution for 2 h. Subsequently, samples were washed twice with 0.1 M acetate buffer, incubated with 0.5% uranyl acetate for 30 min and rinsed two times in 0.1 M acetate buffer. Kernels were dehydrated in an acetonitrile series (30–100%) before embedding in epoxy EMbed 812 resin and polymerizing (48 h, 60 ◦C). A Reichert Jung Ultracut E microtome (Leica Microsystems, Washington, DC, USA) was used to obtain semithin and ultrathin sections. These sections were stained with Richardson blue [22] and examined at 20× and 100× by light microscopy in an Olympus BX41 microscope (Olympus, Allentown, PA, USA).
