Levamisole usage for the block of intestinal alkaline phosphatase in immunohistochemical staining

Abstract

Introduction. Immunohistochemical staining by an indirect method with a chromogenic label requires en-zymes, among which is alkaline phosphatase (AP). AP is also present in human tissues. It can break down the molecules of the immunohistochemical substrate, which leads to significant background staining. It is necessary to block endogenous enzymes prior to immunohistochemical staining to reduce this effect. One of the ways to block endogenous alkaline phosphatase is to use levamisole solutions. The aim of the study was to describe the technique of levamisole use to block the alkaline phosphatase intestinal form during immunohistochemical assays. Materials and methods. This article provides the calculations for 0.001M working levamisole solution preparation from a 10% officinal veterinary levamisole hydrochloride produced by Livisto Invesa Industrial Veterinaria S. A., Spain. The inactivation of AP intestinal form was checked by a reaction with one marker (PDGFRb) and two markers (FAP and SMA) on the colon cancer specimens. We used the Abcam ab210061 DoubleStain IHC Kit: M&R on human tissue (HRP/Green&AP/Red, Great Britain) according to the method recommended by the manufacturer with some changes to identify two markers on the same slide. Results. During the immunohistochemical assay, a complete absence of background staining and a bright contrast reaction with antibodies (both with one and two in the same section) using 1mМ levamisole was achieved. It indicates sufficient inactivation of the AP intestinal isoform in the colon cancer specimens. A comparative cost-benefit analysis for one slide using ready-to-use commercial blocking reagents and officinal levamisole solution shows a significant economic advantage of the latter. Conclusion. The high reaction quality and the palpable economic profits open up opportunities for using 1mM levamisole solution for immunohistochemical studies in laboratory practice and research work. Keywords: levamisole, immunochistochemistry, alkaline phosphatase