After intensive washing, samples diluted in Sorensen buffer were fixed with 2% glutaraldehyde.
For intracellular staining, 100-μL aliquots of the purified cell populations, suspended at 1 × 10 cells/mL in BSA stain buffer, were fixed with BD Cytofix BD Biosciencess) for 30 minutes on ice, washed twice as above, and then permeabilized with Perm Buffer III (BD Biosciences) for a further 30 minutes on ice.
After washing two times with the FACS washing buffer, cells were fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry.
After washing with FACS buffer, cells were fixed with 1% (weight/volume) paraformaldehyde for 30 min and then stored in the dark until analyzed by flow cytometry.
After washing with FACS (Fluorescence Activated Cell Sorter) buffer, cells were fixed with 4% paraformaldehyde and analyzed on a FACS Calibur using Cell Quest and FlowJo 7.6.1.
After washing with 0.1 M cacodylate buffer, the cells were fixed with 1% OsO4 in the same buffer, stained with 1% uranyl acetate in 0.1 M maleate buffer (pH 5.2), dehydrated in ethanol, and embedded in Poly/Bed 812 (Polysciences, Warrington, PA).
After washing with wash buffer, the cells were fixed with 4% paraformaldehyde, and mounted onto slides in Aqua PolyMount (Polysciences, Inc., Warrington, PA, USA) supplemented with 1 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Roche, Basel, Switzerland).
For intracellular cytokine staining, cells were fixed with Fixative Buffer, followed by permeabilisation with flow cytometry buffer containing 0.5% saponin (Sigma-Aldrich).
Fibroblasts washed with phosphate buffered saline (PBS) were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 (w/v) for 5 min and washed three times with PBS.
Red blood cells were lysed with RBC lysis buffer then the cells were fixed with 0.1%paraformaldehyde and analyzed on a BD LSRII 8-color analyzer with FloJo software.
Islets freshly isolated or collected after incubation in KRB buffer as described above were fixed with 4% formaldehyde, permeabilized with 5% Triton X-100, and unspecific sites blocked with 5% Normal Donkey Serum (Jackson Immunoresearch Laboratories Inc, West Grove, PA, USA).
