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Stability of Hormone Receptors with Fixation: Implications for Immunocytochemical Localization of Receptors^*

Department of Physiology, The University of Manitoba Winnipeg, Manitoba, Canada R3E OW3

Address requests for reprints to: Dr. H. Salih, Protein and Polypeptide Hormone Laboratory, Department of Physiology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Canada R3E OW3.

Abstract

The effects of several fixatives on the PRL receptorhave been examined using slices and particulate fractions from pregnant rat and rabbit liver and rabbit mammary glands. After fixation using picric acid-formaldehyde, the binding affinity for ovine PRL (oPRL) was not substantially affected, while the capacity of the oPRL receptor was reduced from 689 to 420 fmol/mg in the rat liver and from 212 to 143 fmol/mg in the rabbit liver membrane fraction. Similarly, fixation using Lillies' buffered formalin reduced the binding capacity of the oPRL receptor in the rat liver to 410 fmol/mg and, in the rabbit liver membrane fraction, to 153 fmol/mg. Fixation of the 100,000 x g pellet using 10% (vol/vol) unbuffered formaldehyde and 4% paraformaldehyde plus 5% glutaraldehyde destroyed specific oPRL-binding sites, and the latter fixative increased nonspecific binding significantly (P < 0.05). Other fixatives gave intermediate results. Direct exposure of the 100,000 x g pellets to 5% acetic acid in ethanol and 7% formaldehyde plus 3% acetic acid in ethanol also destroyed specific binding sites in pellets prepared from rat liver, whereas pellets from the rabbit liver exposed to 5% acetic acid in ethanol exhibited a significant increase in nonspecific binding (P < 0.05) and a decrease in binding capacity from 212 to 104 fmol/mg in the case of 5% acetic acid in ethanol and to 135 fmol/mg in the case of 7% formaldehyde plus 3% acetic acid in ethanol mixture.

These results indicate the desirability of employing biochemical techniques to determine the stability of hormone receptors to various fixatives before using immunocytochemical techniques to study hormone receptor interactions after fixation.

Footnotes

^* This work was supported by a grant from the MRC of Canada and NIH Grant HD-07843-06.

Received December 15, 1978.