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-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.
McKanna
J A
JA
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Zhang
M Z
MZ
D000818:Animals; D017305:Annexin A1 / Q000378:metabolism*; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D001921:Brain / Q000378:metabolism*; D003681:Dehydration; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D007150:Immunohistochemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013194:Staining and Labeling
D017305:Annexin A1; D000906:Antibodies; D007106:Immune Sera; D011994:Recombinant Proteins