[1] Rapid Tissue Fixation and Extraction Techniques

The objectives of preparing tissue for analysis of metabolite content include freezing the tissue rapidly enough to prevent changes associated with excision or other disturbances of the normal milieu of the tissue; preventing alterations during storage and during manipulation before extraction; and extracting the tissue in order to avoid alteration of metabolites by enzymes or by the extraction medium. The same objectives are sought for assay of enzymes that are transformed from nonactivated to activated states, and vice versa, by drug or hormone action on cells, e.g., phosphorylase kinase, phosphorylase, and glycogen synthase in response to epinephrine. It is essential that the enzymes that may affect cAMP concentration be inactivated before the tissue sample begins to thaw (at approximately -8˚C). This means that dissection and weighing must be done at -15˚C or lower, and that the samples are exposed to this temperature for the minimum time. The safest procedure is to transport samples from the freezer to the preparation area and subsequently to the homogenizer in liquid N₂. Specimen tubes or vials should not be tightly capped, because liquid N₂ trapped in the tubes will cause them to shatter when exposed to higher temperatures.