Aquamount

Freshly cut kidney sections (5 μm, two sections per glass slide) from preserved kidney blocks from mice included in the previous study were air dried for 15 minutes, washed twice with PBS (pH 7.4), blocked with 5% goat serum (Genetex, Irvine, CA) for 30 minutes and incubated with primary antibodies for 1 hour at room temperature. In one set of experiments sheep anti-human/mouse fibrinogen (dilution 1:200) and rat anti-mouse CD41 (dilution 1:100) were used. In another set of experiments, the above anti-fibrinogen antibody plus a rabbit anti-mouse vWF (dilution 1:200) were used. In control experiments, rabbit anti-mouse podocin (dilution 1:1,000) was used. Primary antibodies were prepared in PBS with 5% goat serum. All sections were washed three times for 3 minutes each with PBS/5% goat serum and then incubated with secondary antibodies in PBS/5% goat serum. For secondary antibodies, we used a donkey anti-sheep Alexa 647 (dilution 1:500) for fibrinogen detection; a donkey anti-rat Alexa 488 (dilution 1:2,500) for CD 41 detection; and, a goat anti-rabbit Alexa 488 (1:5,000) for vWF and podocin detection. Sections were washed with PBS/5% goat serum 3 times for 3 minutes each and fixed with Aqua-Mount (Polysciences, Warrington, PA). Control kidney sections lacked primary antibodies.

Mice were deeply anesthetized with phenobarbital and transcardially perfused with 10 ml ice-cold Dulbecco’s PBS (DPBS) followed by 10 ml ice-cold 4% PFA. Brains were removed and immersed in 4% PFA overnight at 4°C. Brains were then washed with DPBS with 0.1% sodium azide at 4°C prior embedding in 2% agarose and making 40 μm transverse sections using a vibratome (Leica Microsystems, Wetzlar, Germany, VT1200). Tissues were mounted on slides using AquaMount (Polysciences, Warrington, PA, United States). Microscopy and imaging were done using the Neurolucida system (MBF Bioscience, Williston, VT, United States) with a Zeiss Axioskop microscope with computer-driven stage and Zeiss MRc camera.

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).