SQSTM1/p62 Antibody #5114

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Immunohistochemistry was performed using EnVision (TM) FLEX Mini kit, according to the manufacturer's protocol (cat. no. K8024, Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Briefly, slides were deparaffinized and hydrated in xylene and a graded series of alcohol. Heat-induced antigen retrieval was performed and endogenous peroxidase activity was blocked using a peroxidase-blocking reagent (as part of the EnVision FLEX Mini kit; cat. no. K8024-DM821, Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Sequentially, the slides were incubated with primary antibodies, HRP-conjugated secondary antibody (as part of the EnVision FLEX Mini kit; cat. no. K8024-DM822, Dako; Agilent Technologies, Inc.), diaminobenzidine and hematoxylin for 30 sec, at room temperature. Primary antibodies included p62 (cat. no. 5114, 1:50 dilution, Cell Signaling Technology, Inc.), Na-K-2Cl cotransporter (NKCC-2; cat. no. AB3562P, 1:200 dilution; EMD Millipore), aquaporin-2 (AQP2, cat. no. 3487S, 1:200 dilution, Cell Signaling Technology, Inc.), and aquaporin-1 (AQP1, cat. no. M00865, 1:50 dilution; Boster Biological Technology). Finally, the slides were analyzed using an optical microscope (Olympus Corporation, Tokyo, Japan).

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