Ultrastructural Analysis of Mast Cells

Preparation of mast cell samples for ultrastructural examination by TEM was performed as described previously (54 (link)). Briefly, isolated peritoneal cells of wild-type and TPC1-deficient animals were resuspended in phosphate buffer (100 mM PO₄, pH 7.2) containing 2.5% glutaraldehyde, and then fixed for 4 h at RT. Fixed cells were subsequently collected, rinsed several times in phosphate buffer only (10 min at 200 rcf), and then postfixed in 1% osmium tetroxide (in H₂O) for 1.5 h at 4 °C. Afterward, samples were dehydrated in an ascending acetone series and embedded in epoxy embedding medium (Sigma-Aldrich). Ultrathin sections contrasted with methylene blue were examined using a Zeiss transmission electron microscope (EM902A). Morphological analyses were performed for 21 wild-type and TPC1-deficient mast cells (four individuals per genotype) using the Image J software (https://imagej.net/Welcome).