Jem 100b

After decapitation, pieces of the SN were taken from rats. The material was fixed immediately, by placing tissue samples in a 2.5% solution of glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Postfixation was performed with 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4). Dehydration was carried out in alcohols of increasing concentration and acetone. Dehydrated samples were embedded in Epon 812 resin. Ultrathin sections 60–70 nm thick were made on an ultramicrotome (Leica EM UC7, Wetzlar, Germany). Sections were contrasted with 1% uranyl acetate and zinc citrate. Samples were scanned using a transmission electron microscope (JEOL JEM-100B, Tokyo, Japan). The total number of mitochondria (nMch) on microphotographs was calculated.

Pigeon pea nodules harvested at 30 DAI were embedded in paraffin as described earlier [67 (link)]. Paraffin-embedded nodules were sectioned with a microtome to a thickness of 10 μm and were stained with hematoxylin and eosin. For ultrastructural studies, 30 DAI old nodules were cut into 2- to 4-mm pieces with a razor blade and immediately fixed in buffered 2.5% glutaraldehyde (pH 7.2; 50 mM sodium phosphate) at room temperature for 4 h. Then, the tissues were washed four times, at 15-min intervals, with 50 mM phosphate buffer (pH 7.2) and were postfixed with 2% aqueous osmium tetroxide for 1 h at room temperature. After several rinses in distilled water, the samples were dehydrated in a graded acetone series and were infiltrated with Spurr’s resin. Thin sections were cut with a diamond knife and collected on uncoated copper grids. Sections were stained with 0.5% aqueous uranyl acetate and 0.4% aqueous lead citrate and viewed with a JEOL JEM 100B (Tokyo, Japan) electron microscope at 100 kV.

Tissue preparation was described in details in our previous paper (21 (link)). Sections were viewed with a JEM-100B (JEOL, Japan) electron microscope. Electron micrographs of oligodendrocytes were obtained at *3300 magnification. Oligodendrocytes were identified by small round or oval nucleus, short cisternae of granular endoplasmic reticulum, polyribosomes and the Golgi complex. Microglial cell bodies had elongated, round or triangular nuclei with electron- dense heterochromatin. Cytoplasm of these cells contains mitochondria, cisternae of endoplasmic reticulum, lysosomes, lipofuscin granules, Golgi complex.