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, proinflammatory cytokine, which can be produced by mast cells and several other cell types. We used a newly developed microwave energy-assisted aldehyde fixation method to prepare purified rat peritoneal mast cells for the postembedding immunogold ultrastructural localization of TNF-alpha. These fixation methods were superior to chemical fixation alone in preserving 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 assistance of microwave energy. TNF-alpha was identified in the large storage granules of rat mast cells. The percent of positive granules in microwave-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 assistance. This location of TNF-alpha in rat peritoneal mast cells suggests that this cytokine can use the regulated secretory route(s) for release from appropriately stimulated rat mast cells into the microenvironment.
Beil
W J
WJ
Department of Pathology, Beth Israel Hospital, Boston, MA 02215.
Login
G R
GR
Galli
S J
SJ
Dvorak
A M
AM
D000818:Animals; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000648:ultrastructure; D007150:Immunohistochemistry; D008297:Male; D008407:Mast Cells / Q000737:chemistry* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008872:Microwaves; D010529:Peritoneal Cavity / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016707:Tissue Fixation / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D014409:Tumor Necrosis Factor-alpha