Rejuvenation Solution

1. Composition: 100 g sodium chloride (NaCl), 5 g sodium sulfate (Na2SO4), 50 mL glycerin, 1,000 mL tap water.

2. Procedure. Sodium chloride and sodium sulfate are dissolved in the water and the solution is filtered. Then the glycerin is added. Just before the jar containing this solution is resealed, a few drops of alcoholic camphor are added. There will be a temporary cloudiness of the solution. For another rejuvenation fluid, see ref. (8).

CARBON MONOXIDE REJUVENATION This method (9) cannot be recommended because of the risk of carbon monoxide poisoning for those who work with the gassing apparatus.

FIXATION FOR ELECTRON MICROSCOPIC STUDIES, HISTOCHEMISTRY, IMMUNOHISTOCHEMISTRY, IN SITU HYBRIDIZATION, AND OTHER SPECIAL LABORATORY PROCEDURES

GENERAL PRINCIPLES For processing autopsy material, the same standard laboratory methods are used that would be applied to biopsy samples. However, compared with the work up for light microscopy (see above), much more attention must be paid to the rapid procurement of the material to keep postmortem changes at a minimum. This is described in Chapter 1 under "Immediate Autopsies for Special Laboratory Procedures Such as Electron Microscopy." Needle biopsies in the immediate postmortem period (Chapter 1) also may provide samples without or with minimal autolytic changes.

TRANSMISSION ELECTRON MICROSCOPY Fixation with 2% glutaraldehyde buffered with Millonig's phosphate buffer at pH 7.4 (see also above under "Formalin Solutions" and under "Glutaraldehyde") has been recommended for transmission electron microscopy but paraformaldehyde or a mixture of 10% formalin solution and 1% phosphate-buffered glutaraldehyde are also suitable fixatives (10). The samples should not exceed 1 mm3 and should not remain in glutaraldehyde for more than 4 d. For long-term storage, the tissue should be embedded and kept in the plastic blocks. One can also place the glutaraldehyde-fixed specimens in buffered formalin solution (see above). For comprehensive discussions of fixatives in electron microscopy, see refs. (11) and (12). If no tissue had been saved for electron microscopy but the need arises at a later time, tissue from the exposed surfaces of formalin-fixed tissue can be obtained and postfixed prior to processing. The same can be done with tissues from paraffin blocks; they are postfixed after the samples were deparaffinized. Obviously, the quality of the electron micrographs suffers considerably under these circumstances. However, depending on the questions at hand, answers still can be obtained in some instances.

SCANNING ELECTRON MICROSCOPY Glutaraldehyde, formalin solution or other fixatives can be used. We have obtained excellent electron micrographs of tissue samples that had been stored in formalin solution for some time. Again, it is most important to keep autolysis at a minimum.

HISTOCHEMISTRY Most histochemical stains can be applied to autopsy tissues that had been obtained after the usual postmortem intervals and that were fixed in formalin solution. If new histochemical applications are used on postmortem material, pilot experiments have to be carried out to determine the effect of postmortem changes. Other aspects of histochemistry and related analytical methods are presented in Chapter 11.

IMMUNOHISTOCHEMISTRY Tissue samples should be obtained as soon as possible after death (see also above under "General Principles"), snap-frozen, or placed in formalin solution. Other authors recommend Bouin's solution of B-5. The sections should be thin enough to permit rapid penetration of the fixative; if formalin is used, they should be removed after 12-18 h and if Bouin's solution is used, 4-6 h suffice. If the tissue sample is thick, a thin slice should be obtained from the exposed tissue surface after the recommended fixation time. If antigens need to be identified that are sensitive to the chemical action of fixatives or if immunofluorescent staining is intended, snap-freezing is the method of choice (13). Other aspects of immunohistochemistry and related analytical methods are presented in Chapter 11.

IN SITU HYBRIDIZATION Buffered formalin, pH 7.0, serves as an excellent fixative for this technology. Fixatives with picric acid (Bouin's) or heavy metals (Zenker's) may interfere with subsequent in situ hybridization. Paraffin-embedded tissue is quite suitable for many commercially available DNA probes.

X-RAY MICROANALYSIS (ENERGY-DISPERSIVE X-RAY MICROANALYSIS) Conventional transmission or scanning electron microscopes may also be able to identify elements such as copper, iron, sulfur, or thorium (14) (elements 5-99 can be identified in this fashion). It is best to use glutaraldehyde-fixed tissue but formalin-fixed tissue can also be used, including, as a last resort, tissue from paraffin blocks or tissues lifted from hematoxylin-eosin stained slides. For further applications, see refs. (12) and (15).

AUTORADIOGRAPHY Postmortem material can be used for the identification and localization of radioactive material. Postmortem changes and choice of fixative have little effect on the quality of the autoradiograms. The demonstration of thorium dioxide contrast medium (Thorotrast) used to be the main application of this method. Presently, electron micrography with energy-dispersive X-ray microanalysis (see above) is a faster and more specific method. For the preparation of auto-radiograms, the reader is referred to appropriate textbooks.

Blood Pressure Health

Blood Pressure Health

Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

Get My Free Ebook


Post a comment