Effects of fixation for immunohistochemistry on tissue concentrations of substance P and somatostatin determined by radioimmunoassay

Concentrations of substance P and somatostatin were measured in preparations of the myenteric plexus (plus longitudinal muscle) of the guinea-pig ileum after fixation and processing for immunohistochemistry and compared with concentrations measured in fresh tissue. Two fixative solutions were used: (i) 4% formalin in phosphate buffer (0.1 M, pH 7.0); and (ii) a mixture of aqueous picric acid with 2% formalin in phosphate buffer (0.1 M, pH 7.0). Tissues were extracted in boiling aqueous acetic acid (2.0 M) either immediately after fixation and processing or after storage for up to four weeks in phosphate-buffered saline (PBS) with or without sodium azide. The concentrations of substance P and somatostatin in these extracts were measured by radioimmunoassay and compared to the concentrations in extracts of fresh tissue. The concentration of substance P in fixed tissue was the same as that found in fresh tissue, whereas the concentration of somatostatin in fixed tissue was half that found in fresh tissue (P<0.01). If the tissue was not subjected to the extensive washing for immunohistochemistry, somatostatin concentrations in fresh and fixed tissue were not significantly different. The concentration of substance P did not change on storage of the fixed tissue in PBS, either with or without sodium azide. The concentration of somatostatin decreased on storage of the fixed tissue in PBS over four weeks to 40% of its original value, but the presence of sodium azide maintained the concentration at 60% at four weeks. Neither fixative solution interfered with the radioimmunoassay except at very high concentrations. Fixation for 24h gave the highest estimates of each of the peptides. It is concluded that fixation can be a useful alternative to freezing for preservation of peptides in tissue for radioimmunoassay.