Reynold s lead citrate

The ultrastructure of mouse trabecular meshwork was examined by TEM followed the procedures described previously^{32 (link)}. Mouse eyeballs were fixed in cold 2.5% glutaraldehyde (EM were then post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences) with 1% potassium ferricyanide (Fisher). They were dehydrated through a graded series of ethanol baths and embedded in Epon (made from dodecenyl succinic anhydride, nadic methyl anhydride, Scipoxy 812 Resin, and 2,4,6-tris(dimethylaminomethyl) phenol (Energy Beam Sciences)). Semi-thin (300 nm) sections were cut on a Reichart Ultracut, stained with 0.5% toluidine blue (Fisher), and examined under a light microscope. Ultrathin sections (65 nm) stained with uranyl acetate (Electron Microscopy Sciences) and Reynold’s lead citrate (Fisher) were examined and photographed at 80 kV on a Jeol 1011 TEM.

For standard (2D) electron microscopy imaging, 80-nm sections were cut from the embedded sample blocks on a Reichert Ultracut-E microtome (Leica Microsystems, Vienna) using a diamond knife (Diatome, Biel, Switzerland) and were collected on uncoated 100- and 200- mesh copper grids (hexagonal or square meshes; Ted Pella, Redding, CA, USA). The sections were post-stained with 1% uranyl acetate (Canemco & Marivac, Gore, QC, Canada; pH not recorded) and Reynolds lead citrate (Fisher Scientific Company, USA; pH not recorded) for 12 and 6 minutes, respectively. Electron micrographs at various magnifications were obtained with a Hitachi 7600 transmission electron microscope (Hitachi High Technologies, Japan) at 80 kV.
Lysosomes in these images were identified according to their well established appearance and features: they are bound by a single lipid-bilayer membrane, with a granular, more or less uniform luminal matrix that is more electron dense than the surrounding cytosol. Secondary lysosomes may also contain less granular structures within the finer matrix. Moreover, lysosomes are normally distinguishable from endosomes by their larger size, hence we set a threshold “diameter” of > 200 nm for acceptance of a lysosome, below which all vesicles were excluded.

Transmission electron microscopy (TEM) was used to evaluate the ultrastructure of the TM as described previously (Yun et al., 2014 (link)). After removing the iris, the limbus tissues (n = 3) from each group were fixed in Karnovsky’s fixative and divided into quarters of each tissue. Subsequently, the tissues were dehydrated and embedded in Epon and 65 nm Ultrathin sections were cut, stained with uranyl acetate (Electron Microscopy Sciences) and Reynold’s lead citrate (Fisher). Sections were photographed at 80 kV on a Jeol 1011 TEM for analysis. For evaluation of ER size, the boundary of ER on each TEM image was delineated and ER region was colored by photoshop (Adobe). Then, the area and perimeter of the ER was calculated by Image pro plus (Media Cybernetics). The ER size was displayed as ER area/ER perimeter (nm²/nm).