Evaluation and optimization of the immunohistochemistry antigen retrieval methods on mouse decalcified joint tissues

Abstract

To evaluate and optimize antigen retrieval (AR) methods for the detection of NF-κB, interleukin-1β, interleukin-6, interferon regulatory factor 5 and matrix metalloproteinase-3 expression in mouse joint tissue using immunohistochemistry (IHC). This study evaluated the differences in several AR methods, including pressure cooking (PC), microwave treatment (MW), water bath cooking (WB), trypsin retrieval (TYR), improved water bath cooking (IWB), and enhanced alkaline pH of IWB (EIWB), in tissue integrity maintenance and IHC staining specificity. The AR methods of TYR and IWB maintained the integrity of the tissue to a great extent. The PC, MW, WB, and EIWB resulted in tissue detachment and were not appropriate for subsequent IHC staining. TYR maintained better tissue morphology and staining intensity than IWB, while the IWB retrieval method had a higher percentage of IHC-positive staining and a less nonspecific background. Both the TYR and IWB AR methods were suitable for IHC staining and could effectively resolve the challenges of tissue detachment, insufficient staining, and nonspecific background in paraffin sections of mouse decalcified joint tissues.