Aging Aging

232 Miquelcontrast, washing with buffer avoids a sudden, drastic change in the environmentof the tissue specimens. Because cellular membranes of aldehyde-fixed samplesmaintain in part their selective permeability, a significant decrease in the osmolarityof the washing solution would result in a marked ballooning of cellularorganelles, particularly mitochondria.24. Washing should not be longer than necessary, as the crosslinks caused by glutaraldehydeare potentially reversible and therefore long washings may affect thefinal appearance of the fine structure.25. Solution changes (during fixation, washing, dehydration, and infiltration) shouldbe carried out with speed to avoid drying of the samples. The original vial servesto perform fixation up to the transfer of the specimens to rubber molds, forembedding.26. Because of the hypotonicity of the water in which osmium tetroxide is dissolved,animal tissues exhibit a marked and rapid swelling when they are immersed inthe final buffer-diluted 1% solution of this reagent. However, this swelling isneutralized by the shrinkage occurring during dehydration.27. Rapid osmium tetroxide penetration lasts up to 1 h and reaches a depth of about0.6 mm. After this period, the fixed outer layers of the tissue samples resist deeperpenetration of the fixative and the concentration of osmium tetroxide in solutiondecreases. Consequently, the rate of penetration of the fixative slows down and ittakes 2 h to fix throughout a tissue sample of 1 mm thickness. Osmium tetroxideis unable to make all cellular components insoluble in water and extraction ofcellular products (proteins) may occur during prolonged fixation. Thus, short fixationtimes are more suitable, considering that the fixation time can be increasedwhen osmium tetroxide is used in postfixation.28. Provided that the duration of each step is long enough to accomplish a gradualreplacement of water by the solvent, dehydration should be as short as possible tominimize shrinkage and extraction of cellular constituents. A rapid dehydrationcauses striking osmotic changes resulting in distortion of the structures. The dehydrationtime required will depend on the size and type of the tissue specimen.29. Even in fixed tissue samples, the possible presence of soluble proteins is to betaken into account and the concentration of ethanol at the start of dehydrationshould be high enough to denature (and insolubilize) these proteins.30. Because of the high volatility of propylene oxide, the vials containing the thirdmixture (1:3, overnight) must be sealed carefully with a stopper and parafilm.31. Residual propylene oxide can be completely eliminated from tissue samples bystoring for 2–4 min the open vials with 100% embedding medium into an ovenprewarmed at 60°C for subsequent polymerization.32. To keep the trimmed block firmly attached to its holder, the block should notextend more than 2–3 mm from the front edges of the holder jaws. The evidentresults of a loose block are sections of uneven thickness, alternation of thick andthin sections, or even total failure of sectioning.33. Trimming of the block should be performed avoiding sharp, deep cuts. To obtainthe form of a short pyramid, the block should be trimmed by short cuts and its tip