<abstract xml:lang="en" xmlns="http://www.w3.org/1999/xhtml"><p>To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed <italic>in vivo or in vitro</italic>. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed <italic>in vivo</italic>, tissue fixed <italic>in vitro</italic> exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.</p></abstract>

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.

Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Institute for Medical Research and Occupational Health, Zagreb, Croatia

Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan

Archives of Industrial Hygiene and Toxicology

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or...

Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Online veröffentlicht: 27. März 2009

Seitenbereich: 7 - 17

DOI: https://doi.org/10.2478/10004-1254-60-2009-1895

Schlüsselwörter
imunocitokemija, imunofluorescencija, kriostatski naresci, membranski prijenosnici, proksimalni kanalići, stanična membrana

This content is open access.

Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Online veröffentlicht: 27. März 2009

Seitenbereich: 7 - 17

DOI: https://doi.org/10.2478/10004-1254-60-2009-1895

Schlüsselwörterimunocitokemija, imunofluorescencija, kriostatski naresci, membranski prijenosnici, proksimalni kanalići, stanična membrana

This content is open access.

Schlüsselwörter
imunocitokemija, imunofluorescencija, kriostatski naresci, membranski prijenosnici, proksimalni kanalići, stanična membrana