Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 48, Issue 3, Pages 355-362Publisher
HISTOCHEMICAL SOC INC
DOI: 10.1177/002215540004800305
Keywords
immunohistochemistry; bromodeoxyuridine; antigen retrieval; enzyme digestion; formalin; methacarn; fixation; image analysis; microwave
Categories
Funding
- DIVISION OF CANCER PREVENTION AND CONTROL [N01CN075011] Funding Source: NIH RePORTER
- NCI NIH HHS [NCI-CN-45029-72, CN-75011-72] Funding Source: Medline
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Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are;conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn far 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.
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