In a series of neurohistochemical experiments the effect of aldehyde fixation upon the detection of horseradish peroxidase (HRP) was examined. These experiments demonstrated that: (a) Increments in fixation of as little as 1 hr significantly decreased the number of labeled neurons; 12-hr fixation abolished HRP activity in many neuronal populations and significantly reduced the apparent size of the injection site. (b) This negative fixation effect was greatest where the HRP concentration was low (e.g. in small, lightly labeled neurons) but was still evident in areas of high concentration (e.g. large, heavily labeled neurons). (c) This effect was also most prominent when a less sensitive diaminobenzidine histochemical procedure was employed but was still apparent with a more sensitive benzidine dihydrochloride procedure. (d) Immersion of the brain in fixative after perfusion produced a greater attenuation of HRP activity in more superficial areas. (e) Immersion of the brain in buffer to terminate fixation produced a prolonged and unpredictable gradient of fixation. (f) Excess, unbound fixative inhibited the histochemical reaction per se and had to be removed from the tissue but prolonged washing did not resurrect enzyme activity which was lost by fixation. To obviate these problems and optimize HRP enzyme activity a new perfusion-fixation procedure was developed. It entails 30 min fixation by perfusion which is terminated by a subsequent 30 min perfusion with cold sucrose-buffer to wash out unbound fixative. This allows the tissue to be processed immediately, produces a uniform and morphologically adequate fixation, and minimizes the negative effects of fixation on HRP enzyme activity.
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