Em ac20 ultrastain kit 2

To investigate the presence of protein aggregates on the ultra-structural level, transmission electron microscopy (TEM) was carried out: cell pellets of PPP1R21-mutant and two controls fibroblasts were fixed with 2.9% glutaraldehyde/0.4 M phosphate buffered saline (PBS) and were processed with a Leica EM TP tissue processor with 1%-osmium-tetroxide and embedded in resin. For electron microscopy, ultrathin sections were contrasted with 3% lead citrate trihydrate with a Leica EM AC20 (Ultrastain kit II) and were examined using a Zeiss EM 109 transmission electron microscope equipped with a Slowscan-2 K-CCD-digital camera (2 K-wide-angle Sharp:eye).
Myelin figures (MF) were analyzed at ultrastructural images (magnification × 4.400 and 12.000). MF were counted in all fibroblasts in which the nucleus was gated.

Samples for TEM analysis were available for 13/15 patients. Small samples were taken from open muscle biopsies, fixed in 4% glutaraldehyde/0.4 MPBS, and processed according to standard procedures. For contrast in transmission electron microscopy (TEM), ultrathin sections were treated with 3% lead citrate-3H₂0 with a Leica EM AC20 (ultrastain kit II) and examined at a Zeiss EM109 TEM, equipped with a sharp eye digital camera. Muscle pathology was scored from normal or absent (0) to strong (3), and myofibrillar disintegration, Z-band alterations, and glycogen deposits were analyzed in longitudinal sections at magnification 12,000×. Mitochondrial pathology and tubuloreticular deposits (TIR) in endothelial cells of endomysial capillaries were analyzed in cross sections at magnification 12,000×. At least 10 endomysial capillaries in each specimen were analyzed. The presence of deposits was rated from 0 to 3. Myonuclear actin inclusions were studied in selected cases with ASyS (P4, P14, P6). At minimum, 200 nuclei were analyzed in each specimen.

Small primary cardiac tissue samples were fixed with 6% glutaraldehyde/0.4 M PBS and processed with a Leica EM TP tissue processor. CBs were fixed with 3% glutaraldehyde/0.1 M Cacodylate buffer. The cell pellets were processed by hand according to the automated tissue processor. For electron microscopy of the small cardiac tissue samples and CBs, ultrathin sections were contrasted with 3% lead citrate trihydrate with a Leica EM AC20 (Ultrastain kit II) and were examined using a ZEISS EM 109 transmission electron microscope equipped with a Slowscan-2K-CCD-digital camera (2K-wide-angle Sharp: eye), while CBs were imaged using a Hitachi H-7100.