Immunohistochemical localization of lipocortin 1 in rat brain is sensitive to pH, freezing, and dehydration.

McKanna JA, Zhang MZ
J Histochem Cytochem. 1997 45 (4): 527-38

PMID: 9111231 · 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.

MeSH Terms (13)

Animals Annexin A1 Antibodies Antibody Specificity Brain Dehydration Freezing Hydrogen-Ion Concentration Immune Sera Immunohistochemistry Rats Recombinant Proteins Staining and Labeling

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