0 1 m sodium cacodylate buffer ph 7

Animals (Sms1^-/- n = 2, SMS1^+/+ n = 2, age: 12 weeks) were transcardially perfused with 50ml ice cold PBS, prior to perfusion with 70ml of fixing solution (2.5% PFA, 2.5% glutaraldehyde in PBS). Testes and epididymides were removed and cut into 1mm³ cubes, which were post fixed in 2.5% glutaraldehyde containing 0.1M sodium cacodylate buffer (pH 7.4) at 4°C over night.
Tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide (Dalton, 1955), dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and cured for 24 hours at 60°C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).

Animals (a subset of 5 Irp2KO and 4 WT mice) were transcardially perfused with 50 ml ice cold PBS, prior to perfusion with 70 ml of fixing solution (2.5% PFA, 2.5% glutaraldehyde in PBS). Different brain regions were removed and cut into 1 mm³ cubes, which were postfixed in 2.5% glutaraldehyde containing 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C overnight. Tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide, dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and baked for 24 hours at 60°C. Semi thin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, contrasted with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss EM 10 CR electron microscope, Carl Zeiss NTS GmbH, Oberkochen, Germany).