Immunohistochemistry of GLUT4 in Cryosections

Cryosections for immunohistochemistry have been prepared as described previously^{12 (link)}. Sectioned samples were left 1 h at room temperature to dry, before being fixated in formaldehyde for 5 min. Sections were blocked in PBS (3% BSA) for 30 min, washed and subsequently incubated with GLUT4 (1:1000 in PBS (1% BSA), provided by A. Schürmann, DIfE Potsdam) for 1 h at room temperature. After washing, sections were incubated with a biotinylated secondary antibody (1:200 in PBS; Dianova, Germany) for 30 min, washed and incubated with Streptavidin-Cy3 (1:200 in PBS; Dianova, Germany) for 30 min. Subsequently the sections were washed and incubated with Hoechst (1:1000 in PBS; Thermo Fisher, Germany) for 5 min to stain for nuclear positioning, washed and mounted on slides using Aqua Mount (Polysciences, Germany).