title: A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections.
abstract: Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30 min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10 mM sodium citrate, pH 6.0) was used for PCNA, and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method.
authors.affiliation: Department of Histology and Embryology, Basic Medicine College, China Medical University, No. 77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning, 110122, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Gu
authors.affiliation: Department of Histology and Embryology, Basic Medicine College, China Medical University, No. 77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning, 110122, China.|Department of Histology and Embryology, Shenyang Medical College, Shenyang, Liaoning, 110034, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Cong
authors.affiliation: Department of Histology and Embryology, Basic Medicine College, China Medical University, No. 77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning, 110122, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Histology and Embryology, Basic Medicine College, China Medical University, No. 77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning, 110122, China.
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Tian
authors.affiliation: Department of Histology and Embryology, Basic Medicine College, China Medical University, No. 77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning, 110122, China. zhaixy@mail.cmu.edu.cn.
authors.forename: Xiao-Yue
authors.initials: XY
authors.lastname: Zhai
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D000941:Antigens / Q000276:immunology* / Q000302:isolation & purification*; D006358:Hot Temperature; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008815:Mice, Inbred Strains; D008872:Microwaves; D016612:Paraffin Embedding; D013194:Staining and Labeling; D016707:Tissue Fixation
chemical_list: D000447:Aldehydes; D000941:Antigens
keywords: Antigen retrieval; Fixative; Immunohistochemistry; Microwave heating