Aging Aging

228 Miquelsteps to carry out a conventional double staining with uranyl acetate (ethanolsolution) and lead citrate are:1. An ethanol atmosphere is produced within the Petri dish by placing a piece offilter paper wetted with ethanol 50% at one side of the staining surface.2. A small volume of uranyl acetate is drawn up from below the surface of the stainingsolution with the aid of a clean pipet and a small drop is placed on the paraffinsurface (Note 38).3. The grid to be stained is wetted in distilled water (Note 39) and then floated onthe drop of uranyl acetate, where it should be left from 30 s to a few minutesdepending on whether or not it must undergo multiple staining.4. The stained grid is then thoroughly washed by immersion in a small beaker containinga 50% aqueous solution of ethanol for at least 5 min. Washing can also becarried out by holding the grid with tweezers and dipping and shaking it in thesolution, in the beaker (Note 40).5. The washed grid is then dried by placing it on a filter paper, taking care that thesection side does not touch the paper.6. Steps 1–5 can be repeated to stain the same grid with lead citrate. However, asregards step 1, a carbon dioxide free atmosphere must be generated within thePetri dish by placing a small amount of NaOH pellets at one side of the stainingsurface. Washing should be carried out using twice-distilled water. Usually, theduration of the lead staining (5–15 min) is longer than that with uranyl acetate(Note 41).3.6. Quantitative Mitochondrial StudyA mere qualitative evaluation by electron microscopy of tissue samples isnot enough to document reliably the age-related changes. Therefore, quantitativefine-structural determinations must be performed. With the introduction ofthe dissector procedure (8–10), the methods of estimating subcellular particleand organelle number and size have been changed, with a considerable improvementof the reliability of the findings by moving from assumption-dependentto assumption-free methods (Note 42). Thus, suppositions about particle size,shape, and orientation have been replaced by design-based random sampling,which is a well grounded and reliable procedure to carry out quantitations. Itmust be pointed out that, because these unbiased stereological techniques haveentered into common usage during the last decade, most published morphometricdata have been obtained without the use of the dissector. This does notmean that useful information was not obtained. Thus, to cite an example, in ourprevious studies on the ultrastructure of nerve cell terminals in aged rats, themorphometric techniques in use prior to the dissector method enabled us tomeasure closely related structural parameters of functional significance. Thus,an estimation of the remodelling process taking place in the synaptic regions ofthe aged CNS was obtained. Namely we found that, while the numerical den-