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

Abstract

The amount and localization of three hormones (histamine, endorphin and triiodothyronine [T(3)]) 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 T(3) 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 T(3) 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 T(3) 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.

Perinuclear localization of biogenic amines (serotonin and histamine) in rat immune cells

“…EDAC is a crosslinking reagent which strongly polymerizes biogenic amines and in doing so, prevents their penetration across biological membranes, so they remain at the original site (Handbook, 2005). Comparing EDAC fixation with conventional paraformaldehyde fixation in the case of immune cells, two magnitude higher amounts of histamine were measured by flow cytometry in the EDACfixed preparations (Csaba et al, 2006b). This means that EDAC fixation affords a chance to observe biogenic amines in situ, where they were not seen before.…”

Histidine decarboxylase (HDC) knock out mouse immune cells have altered expression of ACTH, triiodothyronine and endorphin

“…The remnants of the lysed red blood cells were removed by washings in PBS and the cells were fi xed in 4 % EDAC [19] solution. After that, the cells were permeabilized with 0.1 % saponin.…”

Serotonin content is elevated in the immune cells of histidine decarboxylase gene knock-out (HDCKO) mice. Focus on mast cells

“…EDAC fixation preserves more biogenic amines in the cells than the traditional formaldehyde [17]. As a result, not only can cytoplasmic amines be demonstrated more clearly, but nuclear serotonin and histamine contents were also demonstrated [16].…”

“…Correspondence to: G. Csaba Before labeling, erythrolysis was carried out (using Becton Dickinson FACS Lysing Solution). The remnants of the lysed red blood cells were removed by washing in PBS and the cells were fixed in 4 % EDAC [17] solution. After that, the cells were permeabilized with 0.1 % saponin for 3 min.…”