A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Abstract

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.

Development and Application of an Indirect Immunoperoxidase Assay for the Detection of Duck Swollen Head Hemorrhagic Disease Virus Antigen in Pekin Ducks (Anas Platyrhynchos)

“…32 The optimal pretreatment for AR was microwaving in a citrate buffer solution (0.01 M, pH 6.0) for 10 min, which was consistent with previous reports. 5,13,16,20,25,36 To minimize nonspecific staining, tissue sections were pretreated with 3% H 2 O 2 in methanol for 20 min before microwave AR to eliminate false-positive staining caused by endogenous peroxidase. 8,35 The purity of the primary anti-DSHDV antibody, one of the critical agents in IHC stains, 18 was ensured by purification with sucrose density gradient ultracentrifugation and anion exchange chromatography.…”

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

“…Recent technological advances in microwave irradiation devices have also allowed greater control of the irradiation level, irradiation time, and intermittent microwave irradiation (Kumada et al 2004, Temel et al 2005, 2006; therefore, microwave irradiation should be applicable for many histological techniques including immunostaining and electron microscopy (Cavuşog lu et al 1998, Kahveci et al 2003, Koga et al 2003, Ozawa et al 2004). We established a method for rapid fixation and immunofluorescence microscopy using intermittent microwave irradiation.…”

Microwave-assisted antigen retrieval and incubation with cox-2 antibody of archival paraffin-embedded human oligodendroglioma and astrocytomas

“…This procedure found wide application and was often modified to accommodate specific requirements of different tissues or histopathological procedures. Today, these procedures are applied in a variety of histological protocols, such as, fixation, tissue processing, histochemical and immunohistochemical staining for light and electron microscopy, antigen retrieval, and molecular biology techniques (Ainley and Ironside, 1994;Cavusoglu et al, 1998;Giberson et al, 2003;Kahveci, 1993;Kahveci et al, 1997aKahveci et al, , 1997bKahveci et al, , 2003Kennedy and Foulis, 1989;Kok et al, 1988;Leong and Milios, 1993;Login and Dvorak, 1988;Minbay et al, 2001;Noyan et al, 2000).…”

“…Microwave irradiation has been used to improve tissue fixation, enhance histopathological staining for light and electron microscopy Giberson et al, 2003;Kahveci et al, 1997aKahveci et al, , 2003Temel et al, 2005) and more recently, to improve immunohistochemical staining (Evers and Uylings, 2000;Waremborg and Leroy, 2000). Advances in microwave technology facilitated the immunohistochemical analysis of normal and diseased tissues and permitted the development of new antigen labeling techniques (Kumada et al, 2004;Muňoz et al, 2004).…”