Izvorni znanstveni članak
https://doi.org/10.2478/10004-1254-60-2009-1895
Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney
Hrvoje Brzica
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Davorka Breljak
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Marija Ljubojević
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Daniela Balen
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Vedran Micek
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Naohiko Anzai
; Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan
Ivan Sabolić
orcid.org/0000-0002-2587-9109
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Sažetak
To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fi xation, and either frozen and cryosectioned, or embedded in paraffi n before sectioning. p-Formaldehyde (PFA; formalin) is a common fi xative, 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 fi nd an optimal AR method in cryosections of in vivo PFA-fi xed 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-fi xed 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 fi xed in vivo, tissue fi xed 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 fi xation and AR approach.
Ključne riječi
cell membrane; cryosections; immunocytochemistry; immunofluorescence; membrane transporters; proximal tubule
Hrčak ID:
34842
URI
Datum izdavanja:
27.3.2009.
Posjeta: 2.736 *