Em uc7 cryo ultramicrotome

A Philips CM120 electron microscope (Philips, Amsterdam, The Netherlands) with an accelerating voltage of 120 kV was used for transmission electron microscopy (TEM) analysis. Films were firstly cut at −85 °C with a Leica EM UC7 cryo-ultramicrotome (Wetzlar, Germany) equipped with a diamond knife to obtain ultrathin sections of about 90 nm thicknesses. The samples were placed on Formvar coated grids for observation. Low electron beam intensity and short time of exposure were used for observation to avoid any degradation phenomenon. A quantitative analysis of the size of the dispersed domains was performed thanks to ImageJ software (National Institute of Health, open source).

Immuno electron microscopy was performed at the Biocenter Oulu Electron Microscopy Core Facility. Fresh skin samples from adult mouse tail were fixed with paraformaldehyde (4% in 0.1 M phosphate buffer with 2.5 % sucrose, pH 7.4). After fixation, the samples were immersed in sucrose (2.3M), frozen in liquid nitrogen, and sectioned with a Leica EM UC7 cryoultramicrotome (Leica Microsystems, Vienna, Austria). For immunolabeling, sections on Butvar-coated nickel grids were first incubated in 0.1% glycine-PBS for 10 min followed by blocking (serum, 1% BSA in PBS for 5 min). Primary antibodies, anti-EMB (G7.43.1) and anti-MCT1 (AB1286-1, Millipore), were incubated for 45 min and corresponding secondaries, anti-Rat and anti-Chicken IgG (Jackson Immunoresearch Laboratories Inc. Baltimore, PA, USA), for 30 min followed by incubation with protein A conjugated 10 nm gold (Cell Microscopy Core, University Medical Center Utrecht, The Netherlands) for 30 min. 1% BSA in PBS was used in washing steps and dilutions of antibodies and gold conjugates. The grids were stained with neutral uranyl acetate (UA) and coated with 2% methyl cellulose with 0.4 % UA. Tecnai G2 Spirit 120 kV transmission electron microscope (FEI, Eindhoven, The Netherlands) was used for imaging and Quemesa CCD camera (Olympus Soft Imaging Solutions GMBH, Münster, Germany) for capturing the images.