Improved hepatic and pancreatic localisation of the equine alpha-1-proteinase inhibitor family of serpins using an antigen enhancement technique and a monoclonal antibody

Equine alpha- l-proteinase inhibitor (API) consists of three, occasionally four, serum glycoproteins. This study investigated the immunohistochemical localisation of equine API in paraformaldehyde fixed, paraffin embedded equine tissue samples of liver, lung, stomach, pancreas, jejunum and colon in five horses using affinity purified sheep polyclonal and protein A purified mouse monoclonal antibodies, whose specificities were verified by Western blotting. Exposing tissue sections to boiling citrate buffer greatly enhanced antigen recovery and improved immunostaining with both antibodies, resulting in discovery of novel tissue distribution patterns for the horse. In the horses studied, all hepatocytes showed some degree of cytoplasmic staining, many having perinuclear intense granular inclusions. This finding is contrary to findings in human studies where hepatocytes of Pi MM phenotype have proven difficult to stain for human API, despite evidence at the molecular level suggesting hepatocytes as the major source of serum API. This discrepancy may be due to the use of different tissue fixation and antigen recovery techniques. In all other tissues examined, the distribution of equine API was similar to human studies.