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 (12°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. © National Society for Histotechnology 2012.