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

• Published in: The journal of histochemistry and cytochemistry 1997-04, Vol.45 (4), p.527-538
• Main Authors: McKanna, James A, Zhang, Ming-Zhi
• Format: Article
• Language: English
• Subjects: Animals; Annexin A1 - metabolism; Antibodies - isolation & purification; Antibody Specificity; Brain - metabolism; Dehydration; Freezing; Hydrogen-Ion Concentration; Immune Sera; Immunohistochemistry; Rats; Recombinant Proteins - metabolism; Staining and Labeling
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1177/002215549704500405

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.