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Journal of Histochemistry and Cytochemistry, Vol. 49, 475-482, April 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Antigen Retrieval of Basement Membrane Proteins from Archival Eye Tissues

Cheryl R. Hanna, Margaret J. Springettb, and Douglas H. Johnsona
a Departments of Ophthalmology, Mayo Clinic, Rochester, Minnesota
b Electron Microscopy Core Facility, Mayo Clinic, Rochester, Minnesota

Correspondence to: Cheryl R. Hann, Mayo Clinic, Guggenheim 920, 200 First Street SW, Rochester, MN 55905. E-mail: hann.cheryl@mayo.edu

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage 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 glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.

(J Histochem Cytochem 49:475–482, 2001)

Key Words: antigen retrieval, LR White resin, immunolabeling, basement membrane proteins, archival tissue


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