Influence of storage temperature and high-temperature antigen retrieval buffers on results of immunohistochemical staining in sections stored for long periods

The aim of this study was to evaluate the importance of storage temperature in paraffin-embedded sections stored for a fixed period of 3 years. Sections were stained with immunohistochemically stained and treated with high-temperature antigen retrieval (AR). If staining results were impaired, an alternative and more efficient AR protocol was compared with the first to show possible restoration of the immunohistochemical staining. The material represented tumor tissue from two lung carcinomas and four breast carcinomas prepared in a multitissue sandwich block. Sections were cut, mounted on glass slides, and stored for 3 years at -80, 4, or 20 degrees C, or unmounted at 4 degrees C. Additional new sections were cut from the original block the day before staining. The immunohistochemical antigens and clones included ER(1D5), Ki67(MIB1), p53(DO7), C-erbB2(poly), RB1(G3.245), bcl2(124), E-cadherin(HECD-1), and EGFR(113). The first set of AR protocols compared citrate buffer and heating for 25 minutes with TEG buffer and heating for 25 minutes. The second set compared heating for 25 versus 35 minutes after TEG buffer. Positive area was quantitated with the Image Analysis CAS 200 system. The results showed that the positive area decreased with increasing storage temperature for all antigens except C-erbB2, regardless of the AR protocol. Storage at 4 degrees C gave some decrease in positive area, but the antigen expression could be restored, except for EGFR, by the use of an alternative and more efficient AR protocol.