Micropreparation techniques in quantitative histochemistry - density gradient centrifugation, manual microdissection...

Each quantitative histochemical problem needs its specific method for tissue preparation. In this connection two of the most important preparation methods, density gradient centrifugation and microdissection of freeze-dried tissue slices, are described. Density gradient centrifugation is a very effective procedure for preparative separation of cell particles such as cell nuclei. The details of the preparation of glial and neuronal cell nuclei are described. The in vitro phosphorylation of histone in the chromatin in relation to age is given as a practical example of the quantitative histochemical application to a preparation of cell nuclei. Other techniques of tissue preparation are the manual tissue microdissection according to Lowry and the Laser microbeam preparation. Advantages and disadvantages of both methods are compared. It is shown, that the introduction of Laser microbeam dissection technique, as alternative to manual microdissection, add new dimensions to Lowry's ultramicrochemical methods. One has greater freedom in the choice of the sample size and the number of samples dissected from the same slice. Furthermore, the need for a well-trained person for the preparation is eliminated. The preparation is also considerably less time consuming and easier to perform than the manual free hand preparation. Two quantitative histochemical methods used for the investigation of microdissected tissue samples are described: the gas-chromatography-massfragmentography (GC/MS)-method for determination of transmitters and its metabolites as well as the enzymatic cycling technique of Lowry. The GC/MS-method is explained with an example of noradrenaline and dopamine determination. The enzymatic cycling technique is demonstrated in combination with the Oil-Well-Technique for determination of the NADP-cycle.