Tecnai spirit bio twin transmission electron microscope

Immediately after excision, tissues were immersed in fixative consisting of 3% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). After the initial fixation, samples were post-fixed in 1% osmium tetroxide for 1 h, dehydrated in graded acetone solutions, and embedded in Polybed 812 (Polysciences, Inc.). Ultrathin sections (60–80 nm) were cut on an LKB MK III ultratome and routinely contrasted with uranyl acetate and lead citrate. The sections were examined using a FEI Tecnai Spirit BioTWIN transmission electron microscope (Fei) (6 (link)). Eosinophil circularity was calculated as 4Π(total cell area/squared cell membrane perimeter); a value of 1.0 indicates a perfect circle. The granule content was calculated as (cytoplasm area = total cell area - nuclear area) − (the sum of all granule areas).

Cultured MSC were harvested by 0.05% Trypsin-EDTA and cells were seeded in four-well chamber slides (10.000 cells/well) and allowed to adhered over night. After attachment cells were fixed with 1% glutaraldehyde and stained with crystal violet dye. Pictures were taken and the length and width was recorded for 100 cells per sample using the Visiopharm^TM software (Visiopharm, Hoersholm, Denmark). For visualization, 2 × 10⁴ MSC were seeded into trans well inserts (cat no. 3422, Corning, NY, USA) and was left to adhere over night. Cells were fixed with 1.5% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M Sorensen phosphate buffer for one hour, washed and dehydrated with increasing concentration of acetone. The specimen was embedded in Polybed epoxy resin. The specimen block was sectioned on a Leica EM UC7 ultramicrotome and stained with 4% uranyl acetate and 1% lead citrate. The section was analyzed and photographed using a FEI Tecnai Spirit Bio TWIN transmission electron microscope.