Aging Aging

Damage to Mitochondria 231of the flotation water is primarily responsible for the wetting of the knife facet.The optimal wetting of the knife is obtained when the meniscus is flat (with aknife angle of about 90°). To obtain the most advantageous meniscus level, first aconvex meniscus is developed and then, by withdrawing the water with a micropipetup to an initial drying of the cutting edge, the meniscus becomes concave.16. A freshly prepared solution of uranyl acetate should be used, as the recommended3% solution is close to saturation and gradually a precipitate forms with time.However, in our experience, the solution can be used at least for a couple ofweeks, until it looks slightly cloudy.17. The lead citrate solution can be used concentrated or diluted up to 5× with 0.01 NNaOH according to the contrast to be obtained. To prepare lead citrate, the NaOHsolution should be fresh and carbonate free. If it is tightly stopped, the solutionwill stay stable enough for 6–8 wk. When precipitates appear, the solution mustbe discarded.18. The blood washout by saline should be carried out in a short period of time (2–4min) to minimize any tissue deterioration and ensure the best fixative preservation.19. The temperature of the perfusion solution should be kept as close to body temperatureas possible to prevent vasoconstriction. The pressure head of the solutions(saline and the aldehyde-containing fixative), especially for fixation of CNSstructures, must be equivalent to 130–140 cm of water. This can be easily set upby positioning the perfusion bottles shown in Fig. 1 about 130 cm higher than thebody of the animal to be perfused.20. A fixation solution containing the right amounts of glutaraldehyde and paraformaldehydeis more adequate than a solution containing just one of these aldehydes.This is due to the complementary effects of the two chemicals: formaldehydediffuses rapidly and fixes slowly, whereas the glutaraldehyde action is characterizedby a slow penetration and a rapid fixation of tissues, due to the crosslinkingeffects on the adjacent protein molecules.21. The uniformity of fixation throughout the tissue sample is a good criterion foradequate fixation. The size of the specimen exerts an important influence on thehomogeneity of fixation. As a general rule, the smaller the size of the biologicalsample, the more complete and uniform will be the quality of fixation. With theabove described perfusion procedure, a nearly uniform fixation is obtained. However,the size of the samples should not exceed 0.5 mm 3 , as osmium tetroxidepenetrates little in most tissues. Perfused tissues can be easily cut into thin stripsof about 0.5 mm thickness and then sectioned into smaller pieces before osmiumpostfixation.22. If washing of the tissue samples is improperly carried out, residual glutaraldehydewill react with osmium tetroxide and produce a fine, dense precipitate ofreduced osmium.23. If tissues fixed in buffered solutions are washed with water, dissolution and progressivedisintegration of some unfixed cellular structures takes place when theadded electrolytes (or nonelectrolytes) are removed by washing with water. By