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

Abstract

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.

The kinesin KIF21B participates in the cell surface delivery of γ2 subunit-containing GABAA receptors

“…Before staining, we performed antigen retrieval with citric acid pH 8 (Brzica et al, 2009). Briefly, sections were microwave heated 4 × 5 min in citrate buffer pH 8.…”

Sex-dependent expression of Oat3 (Slc22a8) and Oat1 (Slc22a6) proteins in murine kidneys

“…Kidneys were removed, cut in a few sagittal slices, fixed by immersion in 4% p-formaldehyde overnight, washed with four abundant volumes of PBS, and stored in PBS (ϩ0.02% NaN 3) until used. Further steps, such as cutting of 4-m thick tissue cryosections, optimal antigen retrieval methods for unmasking hidden antigens, blocking with albumin solution, as well as incubation with optimal dilutions of primary and secondary antibodies were described in detail elsewhere (6,24). The optimal antigen retrieval for Oat3 and actin epitopes included microwave oven heating in 10 mM citrate buffer, pH 6, whereas the optimal antigen retrieval method for Oat1 epitopes included pretreatment of cryosections with xylol and graded concentrations of ethanol (steps used for antigen retrieval in paraffin sections), followed by microwave oven heating in 10 mM citrate buffer, pH 6 (6).…”

Role of microwave heating in antigen retrieval in cryosections of the formalin-fixed mammalian tissues

“…The SDS-treatment of cryosections of the PFA-fixed tissues and cultured cells was previously demonstrated as a beneficial technique for unmasking epitopes of several membrane-bound proteins (Alper et al, 1997;Brown et al, 1996;Sabolić et al, 1999). However, using similar cryosections of the rat and mouse tissues, we have recently applied a few harsh antigen retrieval protocols that are commonly used to unmask hidden epitopes in the PFA-fixed, paraffin-embedded sections, and efficiently demonstrated the expression of various transporters in the respective organs (Bahn et al, 2005;Balen et al, 2008;Breljak et al, 2010;Brzica et al, 2009aBrzica et al, & 2009bLjubojević et al, 2004Ljubojević et al, & 2007Yokoyama et al, 2008). In these studies, in preliminary experiments we have always tested a battery of antigenrevealing protocols with each antibody and tissue cryosections until one, a protocol that resulted in the highest immunostaining intensity, was defined and further used.…”

“…However, the treatment of tissue cryosections with sodium dodecyl sulfate (SDS), without heating, has been efficiently used to enhance immunostaining with some antibodies (Brown et al, 1996;Sabolić et al, 1999), thus indicating that unmasking techniques may be beneficial for revealing the antibody binding epitopes also in cryosections. Furthermore, we have recently described that heating cryosections of the PFAfixed rat and mouse organs in a microwave oven can be used to enhance immunostaining with specific antibodies for various transporters of organic anions (Bahn et al, 2005;Breljak et al, 2010;Brzica et al, 2009b;Ljubojević et al, 2004Ljubojević et al, & 2007Yokoyama et al, 2008), glucose (Balen et al, 2008;Sabolić et al, 2006), and sulfate (Brzica et al, 2009a). In order to demonstrate the importance of various unmasking protocols for immunocytochemical studies in tissue cryosections, we have used cryosections of the rat kidney and liver tissues that had been fixed with 4% PFA in vivo, treated them with various antigen retrieval protocols without and with the use of microwave heating, and tested for the intensity of immunostaining of several representative proteins known to reside in the cell membrane (cell adhesion molecule 105 (CAM105), megalin (GP 330 ), Na/K-ATPase, aquaporin 1 (AQP1)), cytoplasm (metallothionein), cell membrane and intracellular organelles (vacuolar H + -ATPase (V-ATPase)), and cytoskeleton (actin, tubulin).…”