Influence of paraformaldehyde and EDAC fixation on the demonstrability of hormones (histamine, endorphin, triiodothyronine) in rat immune cells: An immunocytochemical comparative analysis

The amount and localization of three hormones (histamine, endorphin and triiodothyronine [T3]) was measured in male and female rat peritoneal cells (lymphocytes, mast cells, monocyte-macrophage-granulocyte group [mo-gran]) using flow cytometry as well as confocal microscopy after paraformaldehyde (PFA) or EDAC fixation. In the EDAC fixed lymphocytes and mo-gran of female animals two-magnitude higher levels of histamine were measured after EDAC fixation and one magitude higher in mast cells. The amount of T3 was almost four-fold in lymphocytes and 2.5-4-fold in mast cells and mo-gran. Endorphin content was not altered by the type of fixation. In each cell type in males one magnitude higher levels of histamine and T3 were measured after EDAC fixation and a small, but significant, elevation of endorphin. Confocal microscopy supports the quantitative data. The results show that (1) the fixation with the crosslinking molecule, EDAC, is more suitable for immunocytochemical studies of amino-acid type hormones in immune cells, (2) more histamine and T3 are present in the immune cells than it was supposed previously when studying PFA-fixed preparations, (3) the estimation of the amount of peptide hormones seems to be accurate after PFA fixation, (4) there is a quantitative difference comparing the results of PFA and EDAC fixation between males and females.