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Journal of Histochemistry and Cytochemistry, Vol. 45, 527-538, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Immunohistochemical Localization of Lipocortin 1 in Rat Brain Is Sensitive to pH, Freezing, and Dehydration

James A. McKannaa and Ming-Zhi Zhanga
a Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee

Correspondence to: James A. McKanna, Dept. of Cell Biology, Vanderbilt Univ., Nashville, TN 37232.

Lipocortin 1 (LC1, annexin 1) has received considerable attention as a substrate for protein kinases, as a Ca++- and phosphatidylserine-binding protein, and as a mediator of glucocorticoid anti-inflammatory effects. However, there has been confusion over localization of LC1 immunoreactivity (LC1-ir), which reportedly localizes to neurons and/or to astrocytes or microglia in rat brain. To test whether these contradictory data arise from unusual properties of the antigen, we developed a novel brain slice model to determine fixation and staining variables. The specificity of anti-LC1 sera was ensured by pre-absorption and affinity purification with immobilized recombinant LC1. Specific LC1-ir was detected in ramified microglia of brains perfused with acidified aldehydes and embedded in paraffin. However, commonly used immunohistochemical procedures have unexpected profound effects. LC1-ir was eliminated by fixation with neutral/alkaline aldehydes, by freezing before strong acid-aldehyde fixation, or by staining without partial de/rehydration before the primary serum. The sensitivity of LC1 epitopes to proton and water activities may reflect molecular properties important to LC1's roles in vivo. True LC1-ir was not detected in normal neurons or astrocytes. (J Histochem Cytochem 45:527-538, 1997)

Key Words: lipocortin, annexin, microglia, astrocytes, neurons


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