Antigen retrieval of basement membrane proteins from archival eye tissues

Antigen retrieval (AR) methods can unmask tissue antigens that have been al tered by fixation, processing, storage, or resin interactions. This is part icularly important in the study of archival tissues, because primary fixati ves and storage times may Vary among specimens. We performed an electron mi croscopic study of basement membrane components of the aqueous humor draina ge pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glu taraldehyde. Labeling density was approximately doubled after AR for most f ixatives, with the largest increase for tissues fixed in 4% paraformaldehyd e/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular ma trix labeled; no "new" components were labeled after AR. We conclude that A R in citrate buffer can be used on selected extracellular matrix antigens t o enhance label that would otherwise be lost due to fixation and storage.