Em109 electron microscope

UVB-irradiated mice were lethally anesthetized with pentobarbital (700 mg/kg) and perfused with 4% PFA in PBS. Skin samples were kept overnight in fixation solution, rinsed, and stored in PBS. After washing in 0.1 M cacodylate buffer pH 7.2 at 4 °C, the specimens were treated with 2% OsO₄ for 2 h. After washing, they were stained in 1% uranyl acetate, dehydrated, and embedded in araldite resin. Ultra-thin sections (30–60 nm) were processed with a diamond knife and placed on copper grids. Transmission electron microscopy was performed using an EM109 electron microscope (Zeiss, Oberkochen, Germany).

In ultrastructural analysis, samples were fixed in Karnovsky-lto solution (4% PFA, 5% glutaraldehyde, and 0.05% picric acid in cacodylates buffer with pH: 7.2–7.4) (Ito, 1968 ) during 24 h at room temperature (RT). For post-fixation we used 2% aqueous osmium tetroxide for 1 h; samples were then treated with a 1% aqueous solution of uranyl nitrate. After rapid dehydration with 30%–100% gradual acetone, tissues were embedded in epoxy resin. Sectioning was done with a diamond knife on an RMC ultramicrotome. Semithin 350 nm thick sections were stained with 1% toluidine blue (TB) for the study of apoptotic and/or necrotic features with a light microscope. Also, ultra-thin (70 nm) sections were collected on 200-mesh copper grids without a supportive substrate. Sections were counterstained with uranyl acetate and lead citrate and analyzed on a Carl Zeiss EM109 electron microscope.