1200 ex 2 transmission electron microscope

Animals were transcardially perfused with 20mL of PBS. The left hemisphere was dropped fixed in 4% PFA for immunohistochemistry analysis. A 1mm slice cut from the medial side of the right hemisphere was fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Ted Pella Inc., Redding, CA) in 100mM cacodylate buffer, pH 7.2 for 2 hours at room temperature and then overnight at 4°C. Samples were washed in cacodylate buffer and postfixed in 1% osmium tetroxide (Ted Pella Inc.) for 1 hour. Samples were then rinsed extensively in dH₂0 prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 hour. Following several rinses in dH₂0, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX II transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA). 5000X and 15000X images of corpus callosum were taken for analysis. G-ratio (diameter of axon divided by the diameter of axon plus myelin) was quantified using MyelTracer Software [https://github.com/HarrisonAllen/MyelTracer].

G-actin (4 µM) was incubated in 0.4 mM ATP, 2 mM Tris, pH 7.5, 0.2 mM CaCl₂, 0.01% NaN₃, and 0.5 mM DTT for 10 min on ice. Polymerization buffer (20X) was added to G-actin to reach a final concentration of 1X (100 mM KCl, 25 mM imidazole, pH 8.0, 2 mM MgCl₂, 1 mM EGTA, and 0.1 mM ATP), and actin was polymerized for 30 min at room temperature. F-actin was kept at 4°C for several hours during TEM imaging. From the same batch of prepared F-actin, all TEM samples (including the actin-only control) were prepared. Wild-type Tpm1.1 (1.25 µM) or 2 µM Tpm1.1[R21H] was added to F-actin and the mixture was incubated at 4°C for 10 min. Lmod2 (0.9 µM) was added to F-actin in the absence or presence of Tpm1.1 and incubated at room temperature for 10 min.
To visualize complexes of F-actin with Lmod2 and with or without bound Tpm1.1, aliquots (7 µl) were applied to glow-discharged carbon-coated grids (Electron Microscopy Sciences, Hatfield, PA) and stained with 2% uranyl acetate. The grids were examined in a JEOL 1200 EXII transmission electron microscope (JEOL USA, Peabody, MA) under regular dose conditions at an accelerating voltage of 70 keV.
Pellet samples from cosedimentation assays were run on 10% polyacrylamide SDS gels. The gels were scanned using ChemiDoc XRS (Bio-Rad, Hercules, CA) and quantified using the National Institutes of Health ImageJ software.

Whole bladders of WT and Ifrd1^−/− mice were fixed using 2% glutaraldehyde and 3% paraformaldehyde in 0.1 M sodium cacodylate. Samples were then washed a total of three times in sodium cacodylate buffer and post-fixed in 1% osmium tetroxide for one hour, and then stained in 1% uranyl acetate for an hour, before being rinsed, dehydrated, and subjected to critical point drying. Bladder samples were then gold-coated and viewed using JEOL 1200 EX II Transmission Electron Microscope (JEOL, USA). For quantification of MVB, lysosomes, and mitochondria, images of superficial cells were taken at 2500x. The number of MVBs, cargo-containing MVBs, mitochondria, and lysosomes were quantified, and the amounts were reported as per 100μm² surface area examined. (n= 50-150 TEM sections from four individual mice).