IMMUNOHISTOCHEMICAL LOCALIZATION OF LIPOCORTIN-1 IN RAT-BRAIN IS SENSITIVE TO PH, FREEZING, AND DEHYDRATION

Lipocortin 1 (LC1, annexin 1) has received considerable attention as a substrate for protein kinases, as a Ca++- and phosphatidylserine-bind ing protein, and as a mediator of glucocorticoid anti-inflammatory eff ects. However, there has been confusion over localization of LC1 immun oreactivity (LC1-ir), which reportedly localizes to neurons and/or to astrocytes or microglia in rat brain. To test whether these contradict ory 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 aff inity purification with immobilized recombinant LC1. Specific LC1-ir w as detected in ramified microglia of brains perfused with acidified al dehydes and embedded in paraffin. However, commonly used immunohistoch emical procedures have unexpected profound effects. LC1-ir was elimina ted by fixation with neutral/alkaline aldehydes, by freezing before st rong acid-aldehyde fixation, or by staining without partial de/rehydra tion before the primary serum. The sensitivity of LC1 epitopes to prot on 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.