Aging Aging

Damage to Mitochondria 233should not be too thin nor too long. Otherwise, it will vibrate at high frequencywhen hitting the cutting edge.34. Before collecting the sections, the compression due to the impact of the blockwith the cutting edge of the knife can be relieved by exposing the sections verybriefly to the vapors of a strong solvent such as chloroform.35. The grid should be pressed gently over the floating sections with the aim ofdepressing (not breaking) the water surface. As soon as the grid reaches across aribbon, the sections stick firmly to the grid along with a drop of water. Becausethe electrostatic charges on the grid surface are responsible for the attraction ofthe ribbon, rinse the grid with acetone just prior to use, to reduce the charge andget a better control in orienting and centering the ribbon.36. The most frequent mistake in making sections for electron microscopy isrepresented by periodic variations in the contrast of a section (commonly termed“chatter”). Usually, this failure is not detected during sectioning, but only whileviewing the section at the electron microscope when it is too late to correctthe mistake. Usually, “chatter” is due to vibrations of microtome parts that includethe cutting edge, the specimen block, and the microtome arm. However, someother causes may be involved, for example, too fast cutting speed, too large and/orirregular block face, specimen or knife loosely held, too hard or too soft specimenblock, dull knife, and incomplete polymerization of the epoxy resin.37. Contrast in an electron micrograph depends essentially on the density andthickness of the different parts of the section. By combination with elements ofhigher atomic weight (osmium, uranium, and lead), some cellular structures aremade denser than their surroundings.38. The first one or two drops of stain from the pipet are discarded to avoid anycontamination. The drop used should be small enough to allow the grid to floaton it instead of sliding down to its sides.39. Wetting of the sections decreases the risk of contamination due to extensivestain–air contact.40. To remove any excess of uranyl acetate, a quick rinse in 0.5% tartaric acid can beperformed after washing in a 50% aqueous solution of ethanol. This step must becarried out very rapidly (2–3 s!) and it must be followed by repeated washings indistilled water.41. Keep the staining time as short as possible because there is a risk of overstaining,which results in an overall increase in contrast with poor differentiation of cellularstructures.42. The dissector method is designed to identify particles seen in one section (“lookupsection”) but not in a following one (“reference section”), and serves to evaluatethe number of particles per unit volume (numerical density: Nv). Serialsections are needed to perform counting of particles or organelles. For instance,the mitochondria present in a discrete area of the reference section, but not in thelook-up section are counted (Fig. 3, arrows) and referred to the height of eachdissector which takes into account the section thickness (further details in refs.11–13).