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Lee, Eung; Yin, Hui; Kear, Megan; Haffajee, Zenobia; Pepperall, Debbie

doi:10.1097/00022744-200209000-00014

Cited by 11 publications

(2 citation statements)

References 17 publications

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“…Harsh antigen retrieval methods, such as delipidation with alcohols, microwave heating in buffers of different pH, and autoclaving, are usually used to recover cryptic epitopes in sections of formalin-fi xed, paraffi n-embedded tissues (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)12). Such techniques have not regularly been used with cryosections.…”

Section: Discussionmentioning

confidence: 99%

“…Most of the previous studies of antigen retrieval methods were performed with sections of paraffin-embedded tissues/cells. Depending on the antibodies used, these methods include treatment with various alcohols (to remove paraffi n), different heating temperatures, pressures, osmolality, and chemical composition of the reagents, pH, and different timing of treatments (5)(6)(7)(8)(9)(10). The pH of reagents seems to play a crucial role in antigen retrieval; pH 8 to pH 9 has been proposed as optimal for starting with a new antigen (9).…”

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confidence: 99%

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

Brzica

Breljak

Ljubojević

et al. 2009

Archives of Industrial Hygiene and Toxicology

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

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