Aging Aging

Damage to Mitochondria 2271. 100% propylene oxide: 20 min (three changes).2. Propylene oxide and embedding resin (3:1): 30 min.3. Propylene oxide and embedding resin (1:1): 30 min.4. Propylene oxide and embedding resin (1:3): overnight (Note 30).5. Embedding resin 100% (Note 31): 30 min.Final embedding is carried out in rubber molds of different size which allowan easy orientation of the tissue samples. Polymerization of the epoxy resin is completedwithin 48 h at 60°C. Polymerized blocks can be sectioned best after 2 d.3.4. Sectioning (Fig. 2)Because sections are cut from a very small portion of the tissue specimens,trimming of the blocks must be carried out. The block is mounted on the blockholderwhich is placed in the microtome (Note 32). Rough trimming of theblock must be performed with a sharp razor blade to remove the excess ofembedding medium around the tissue sample. Too large tissue samples shouldbe also reduced in size to obtain a block face of suitable size. Afterwards, finetrimming is carried out with the aim of obtaining a block tip with smooth andclean sides. In our experience, when making sections from a brain sample, it isusually necessary to examine a semithin section (thickness: 1.5 µm) at the opticalmicroscope to select the area to be viewed at the electron microscope. Whenthe area of interest in the tissue sample is identified, the block is retrimmed toobtain a trapezoid face with parallel lower and upper sides and two slopedsides (Note 33). Because the shortest of the two parallel edges is mounted up,each section will move the previous one from the cutting edge and a straightribbon of sections will be formed (Fig. 2; Note 15). Section collection (Note34) is accomplished by the following steps:1. Hold the edge of a grid with a pair of curved tweezers.2. Bend the edge of the grid by about 100°.3. Lower the grid onto the upper face of the ribbon (Note 35).4. Dry the grid with the sections by placing it on filter paper which absorbs thewater (of course the side of the grid without sections is to be brought into contactwith the filter paper!) (Note 36).3.5. StainingTo obtain a better contrast and resolution, the sections mounted on a grid arestained by the deposition of heavy metal ions, such as uranium and lead (seeNote 37). The simplest method to stain sections is to let the grid (with sectionside down) float on a drop of a staining solution in a Petri dish. The stainingsurface is constituted by the lid of a glass Petri dish partially filled with meltedparaffin which is allowed to cool. This surface must be kept very clean andshould be protected from dust by keeping it covered during staining. The basic