PMID: 413864Jan 1, 1978Paper

Fixation variables in horseradish peroxidase neurohistochemistry. I. The effect of fixation time and perfusion procedures upon enzyme activity

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
D L Rosene, M M Mesulam


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 ...Continue Reading


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