Short time incubation at low temperature for retrieval of some antigens from formalin-fixed and paraffin-embedded tissues

Abstract

Antibody manufacturers often recommend antigen retrieval for formalin-fixed paraffin-embedded tissue sections before immunohistochemical staining. The most commonly used methods involve high temperatures (90–120°C) in buffers. Low-temperature retrieval methods allow the use of tissue flotation baths but their incubation times are often long. This study investigated retrieval of some antigens at low temperature (65°C) for 10 minutes in citrate buffer (pH 6·0) using a tissue flotation bath. Sections were obtained from two tissue specimens, breast and O. volvulus nodules, which were formalin-fixed and paraffin-embedded. The antigens of lactate dehydrogenase, pyruvate dehydrogenase kinase-2, malic enzyme-1, succinate dehydrogenase-B, glucose-6-phosphate dehydrogenase, alpha/beta-hydrolase-4, estrogen receptors (ER), and progesterone receptors (PR) were retrieved at low temperature and in the cases of ER and PR, were also retrieved at high temperature (120°C), using a pressure cooker, for comparison. All the retrievals were carried out in citrate buffer, pH 6·0. The results showed that all the enzymes scored 2+ (i.e. a strong reaction) in staining intensity and the staining for ER and PR were similar for both methods (2+). In immunohistochemical staining, the most important stage is antigen retrieval. The low-temperature antigen retrieval in citrate buffer at pH 6·0 provided staining intensities comparable to those by high-temperature methods. The results indicate that low-temperature antigen retrieval is cost-effective, rapid, and reliable.

Keywords:

• Antibodies
• Epitopes
• Flotation-bath
• Pressure cooker

Although all the chemicals and reagents for this project were purchased solely through the collaborative funding of the Department of Pathology of the University of Ghana Medical School and University of Ghana School of Allied Health Sciences through their local research funds, we would like to thank all the members of staff of the Department of Pathology of the University of Ghana Medical School for allowing us to use the equipments and other minor requirements for this project. We are also indebted to Dr K. Awadzi, the director of Onchocerciasis Chemotherapy Research Centre at Hohoe hospital, Ghana and his staff especially Dr N. O. Opoku and the nurses for donating the Onchocerca volvulus nodules.