Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections.

Abstract

We developed a staining protocol for demonstration of androgen receptor (AR) in formalin-fixed, paraffin-embedded tissue sections. The method is based on the antigen retrieval microwave (MW) heating technique. Results are compared with different types of enzyme digestion pre-treatments. The strongest immunostaining signal and clearest background were obtained by MW heating of dewaxed paraffin sections in 5% urea or citrate buffer solution (pH 6); pure distilled water gave less consistent results. Enzymatic digestion with pepsin (0.05% in 2 N HCl) for 30 min at room temperature, or trypsin followed by pronase, or pronase digestion alone, also produced enhanced staining of AR in some cases, but there was more nonspecific background, and specific reactivity was less intense. The antigen retrieval MW method can be used to demonstrate AR epitope in prostate tissue after fixation in formalin for as long as 7 days. AR immunolocalization was also compared in frozen and paraffin sections processed from the same specimen of prostate carcinoma tissue and was found to be qualitatively and quantitatively similar. This study also provided new information concerning the basic principles of the antigen retrieval MW method that may be helpful in further development of this technique.

Effect of starvation on Fos and neuropeptide immunoreactivities in the brain and pituitary gland of Xenopus laevis

“…Coronal serial sections (7 m) were mounted on poly-L-lysine-coated slides (Sigma, St Louis, MO, USA) and allowed to air-dry, for 16 h at 45°C, deparaYnized, rehydrated, and treated for immunocytochemistry, as described before (Calle et al, 2005a). As to the Fos staining, the antigen retrieval method of Shi et al (1993) was used. After deparaYnization, sections were pre-heated in a microwave oven at 90°C in 0.3% tri-sodium citrate buVer (pH.…”

A Mouse Model for Slowly Progressive Primary Tuberculosis

“…Therefore the accessibility of antigenic epitopes might be limited. Such hindrances may be partially reversed by retrieval techniques like enzyme digestion and microwave oven heating [28][29][30]. To identify a suitable method for antigen retrieval, the effect of pretreatment with enzyme and microwave oven heating was studied in tissues from eight mice in the following three experiments:…”

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…It has been demonstrated that the AR technique is a simple and effective enhancement method for immunohistochemistry on archival tissue sections. Two major advantages of AR-IHC are (a) reduction of the detection thresholds of immunohistochemical staining (increased sensitivity) for a wide range of antibodies, and (b) retrieval of some antigens, such as Ki-67, MIB1, ER-1D5, bcl-2, androgen receptor, RB, CD5, CD8, CD35, CD38, CD25, CD45R, CD34, CD68, CD44, CD55, VCAM-1, CGA7 (smooth muscle-specific actin), CD19, and thrombospondin (Grossfeld et al 1996;Cuevas et al 1994;Cattoretti et al 1993;Gown et al 1993a;Leong and Milios 1993;Shi et al 1993bShi et al ,1996, which are otherwise negative even with other unmasking pretreatment. At present the AR-IHC method is being widely used for the detection of ER, PR, MIB1, p53, bcl-2, pRB, and some CD markers on routinely formalin-fixed, paraffin-embedded tissue sections in daily pathological diagnosis (for review and detailed data see Shi et al 1995b).…”

“…Furthermore, the toxic effect of metal salts, particularly lead salts, is a major drawback. Therefore, several alternative AR solutions were subsequently developed, such as citrate, Tris and other buffer solutions, and urea (Kawai et al 1994b;Taylor et al 1994a;Gown et al 1993a;Leong and Milios 1993;Shi et al 1993b;Gerdes et al 1992), EDTA or EGTA (Morgan et al 1994), glycine-HCl buffer (Imam et al 1995), and periodic acid (Kwaspen et al 1995).…”