ULTRASTRUCTURAL IMMUNOGOLD LOCALIZATION OF TUMOR-NECROSIS-FACTOR-ALPHA TO THE CYTOPLASMIC GRANULES OF RAT PERITONEAL MAST-CELLS WITH RAPID MICROWAVE FIXATION

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional, proinfla mmatory cytokine, which can be produced by mast cells and several othe r cell types. We used a newly developed microwave energy-assisted alde hyde fixation method to perpare purified rat peritoneal mast cells for the postembedding immunogold ultrastructural localization of TNF-alph a. These fixation methods were superior to chemical fixation alone in presenting both the ultrastructural morphology and immunoreactive TNF- alpha in rat mast cells. The percent of TNF-alpha-positive mast cells in samples prepared with microwave-assisted fixation in low (84%) and standard (81%) glutaraldehyde concentrations exceeded that for low (56 %) and standard (15%) glutaraldehyde concentrations without the assist ance of microwave energy. TNF-alpha was identified in the large storag e granules of rat mast cells. The percent of positive granules in micr owave-assisted standard (44%) and low (40%) glutaraldehyde samples was considerably higher than the percent of positive granules in standard (5%) and low (10%) glutaraldehyde-fixed samples without microwave ass istance. This location of TNF-alpha in rat peritoneal mast cells sugge sts that this cytokine can use the regulated secretory route(s) for re lease from appropriately stimulated rat mast cells into the microenvir onment.