An antigen unmasking protocol that satisfies both immunohistochemistry and subsequent PCR amplification.
Immunohistochemical elucidation of many proteins in formalin-fixed, paraffin-embedded tissues requires a prior antigen unmasking treatment, which often damages both the morphology and genetic materials, making subsequent assessments difficult or impossible. This study attempted to develop a method that satisfies both immunohistochemical and genetic analyses. Consecutive sections were made from formalin-fixed, paraffin-embedded breast and other tissues, and a set of four adjacent sections from each case were treated with (1) routine H & E staining; (2) our unmasking protocol; (3) microwave oven irradiation; (4) pressure cooker incubation. After immunohistochemical staining, the tissue in each section was scraped off, or the same cell clusters in four sections were separately microdissected for DNA extraction and PCR amplification. Compared to microwave and pressure cooker methods, our protocol showed the following advantages: (1) a better preservation of the morphology; (2) a substantial reduction of tissue detachments from slides; (3) effectiveness on all antibodies tested, including those requiring enzyme digestion or no prior unmasking; (4) higher PCR yields; (5) larger (higher molecular weight) amplified PCR products. Compared to the routine method on untreated tissues, our method consistently produced a comparable quality and quantity of PCR products. Our protocol, however, takes a longer time to yield results.
Man
Yan-Gao
YG
Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, and American Registry of Pathology, Washington 20306-6000, USA. man@afip.osd.mil
Burgar
Ana
A
D000941:Antigens / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D042282:Microdissection; D008872:Microwaves; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction
D000941:Antigens; D015415:Biomarkers; D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde