Detection of monokines in paraffin-embedded tissues of pigs using polyclonal antibodies

Monokines are glycoproteins, synthesised by macrophages, which exert various effects on the organism. The most important monokines are interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor (TNF)-alpha and IL-6. This paper reports on immunohistochemical techniques developed for the detection of IL-alpha, IL-1 beta, IL-6 and TNF-alpha in fixed and paraffin-embedded pig tissues (spleen, lymph nodes, thymus, liver and kidney). Different fixatives (buffered formalin, acetic formalin, paraformadehyde-lysine-periodate and Bouin solution), and antigen unmasking techniques (permeabilisation with Tween 20, pronase enzymatic digestion and microwave-citrate buffer) were used. We describe different protocols for detection of monokines using polyclonal antibodies against the studied monokines. No signal was obtained with monoclonal antibodies against pig-TNF-alpha and human IL-1 alpha. Bouin solution was shown to be the best fixative for immunohistochemical detection of IL-1 alpha, TNF-alpha, and IL-6, using permeabilisation with Tween 20 as an unmasking antigen method. Acetic formalin was shown to be the best fixative for IL-1 beta detection, not needing antigen retrieval techniques. Macrophages were identified as the main cytokine-producing cells, although other types of cells also stained positively to some cytokines. These techniques represent valuable tools for studies of the pathogenesis of viral and bacterial diseases, and of the immune system of the pigs.