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Evaluation of the Value of Frozen Tissue Section Used as “Golden Standard” for Immunohistochemistry
Author(s) -
Shi ShanRong,
Liu Cheng,
Pootrakul Llana,
Tang Laurie Yee Mae,
Young Andrew,
Chen Ryan,
Cote Richard J,
Taylor Clive R
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a774-a
Subject(s) - immunohistochemistry , frozen section procedure , acetone , antigen retrieval , staining , chemistry , antibody , fixation (population genetics) , pathology , biology , biochemistry , medicine , immunology , gene
To examine the traditional concept that acetone or ethanol‐fixed frozen tissue sections represent the “golden standard” for IHC, we have conducted a study evaluating frozen sections with various conditions of fixation and antigen retrieval (AR). Fresh human tissues were frozen in OCT. An adjacent tissue was fixed routinely in 10% neutral buffered formalin (NBF) and paraffin‐embedded (FFPE). Preparations of human cell lines were processed into frozen and FFPE cell blocks to confirm the IHC results of tissues. Frozen tissue/cell sections were fixed by 6 protocols: acetone 10 min; ethanol 10 min; NBF 30 min, & 24 hrs; NBF + CaCl2 30 min, & 24 hrs. The AR technique was used for all NBF fixed tissue sections. 26 antibodies with ABC kit, DAB, were used to generate the IHC signal. Results: more than half antibodies (16/26, 61.5%) showed identical IHC staining results between acetone‐fixed, and NBF‐fixed tissue sections. Among the remained antibodies, 5 (19.2%) showed better IHC signals following NBF and AR, only 3 antibodies gave better IHC staining results for acetone‐fixed tissue sections. Most cytoplasmic proteins (11/13) showed comparable IHC signal between acetone and NBF fixed tissue sections. For nuclear proteins, NBF‐fixed tissue sections gave better IHC signals than obtained by acetone‐fixed sections. In most cases NBF yielded stronger signal with less background, and better morphology. Conclusion: the data do not support the traditional use of acetone fixed frozen tissue sections as the “golden standard” for IHC. It would be prudent to employ a combination of both acetone and NBF fixed frozen sections, and FFPE tissue sections may serve as the standard for most antigens for IHC. This study is supported by NIH grant 1 R33 CA103455‐03.