Gatan 4 k 2.7 k digital camera

After OVA treatment, WT and PGRN KO mice were anesthetized and the lung was perfused with a fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 hrs. After washing, the samples were postfixed in 1% OsO4 for 1 hour, followed by block staining with 1% uranyl acetate for 1 hour. Samples were dehydrated and embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA). 60 nm sections were cutted and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under Philips CM-12 electron microscope (FEI; Eindhoven, Netherlands) and photographed with a Gatan (4 k × 2.7 k) digital camera (Gatan, Inc., Pleasanton, CA). The preparation of the cell samples for TEM was done with the assistance of Dr. Fengxia Liang at NYU Medical School OCS Microscopy Core.
For immunogold labeling, the mice lung tissue sections were incubated with antibodies against p62, followed by incubation with secondary antibodies labeled with 5 nm gold particles. Sections were observed under electron microscopy.

Transmission Electron Microscopy (TEM) was performed at the NYU Langone Medical Center Microscopy Core. LBOs were fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH7.2) for 2 hours and post-fixed with 1% osmium tetroxide for 1.5 hours at room temperature, then processed in a standard manner and embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA). Semi-thin sections were cut at 1 mm and stained with 1% Toluidine Blue to evaluate the quality of preservation and find the area of interest. Ultrathin sections (60 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under Philips CM-12 electron microscope and photographed with a Gatan (4k ×2.7k) digital camera (Gatan, Inc., Pleasanton, CA).

After OVA treatment, PGRN KO mice were anesthetized and the lung was perfused with fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer (pH 7.2) for 2 h. After washing, the samples were post fixed in 1% OsO4 for 1 h, followed by block staining with 1% uranyl acetate for 1 hour. After dehydration, samples were embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA, USA). 60 nm sections were cut and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under a Philips CM-12 electron microscope (FEI; Eindhoven, Netherlands) and photographed with a Gatan (4 k × 2.7 k) digital camera (Gatan, Inc., Pleasanton, CA, USA).