Tecnaig2 spirit biotwin

Adrenals were fixed in 2.5% glutaraldehyde, 1.25% paraformaldehyde, and 0.03% picric acid in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at RT, washed in 0.1 M cacodylate buffer and post-fixed with 1% Osmiumtetroxide (OsO4)/1.5% Potassiumferrocyanide (KFeCN6) for 1 h. Samples were washed in water twice, in 50 mM Maleate buffer pH 5.15 (MB) once, and incubated in 1% uranyl acetate in MB for 1 h followed by 1 wash in MB, 2 washes in water and subsequent dehydration in an ethanol gradient (10 min each; 50%, 70%, 90%, 2 × 10 min 100%). Samples were then put in propyleneoxide for 1 h and infiltrated overnight in a 1:1 mixture of propyleneoxide and TAAB Epon (TAAB Laboratories Equipment, Aldermaston, England). On the following day, samples were embedded in TAAB Epon and polymerized at 60 °C for 48 h. Ultrathin sections (about 80 nm) were cut on a Reichert Ultracut-S microtome, picked up on to copper grids stained with lead citrate and examined in a JEOL 1200EX Transmission electron microscope or a TecnaiG² Spirit BioTWIN and images were recorded with an AMT 2k CCD camera.

A mixture of 2.5% paraformaldehyde, 5% glutaraldehyde, and 0.06% picric acid in 0.2 mol/L Cacodylate buffer was freshly prepared before use, then the above mixture was diluted 1:1 with dH2O. To Fix primary B cells, 1 million cells were collected and washed with Dulbecco’s Phosphate Buffered Saline (DPBS, 14190, Gibco) one time, remove the residue buffer, then gently add the diluted fixative to the primary B cells. The cells were fixed at RT for 1h. The cells were then post-fixed for 30 min in 1% Osmium tetroxide (OsO4)/1.5% potassium ferrocyanide (KFeCN6), washed in water 3x and incubated in 1% aqueous uranyl acetate for 30 minutes. Samples were then washed twice in water and dehydrated in grades of alcohol (5min each; 50%, 70%, 95%, 2× 100%). Cells were removed from the dish in propyleneoxide, pelleted at 3000 rpm for 3 minutes and infiltrated for 2 hours in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac Canada Inc. St. Laurent, Canada). Samples were subsequently embedded in TAAB Epon and polymerized at 60 degrees C for 48 hrs. Ultrathin sections (about 60nm) were cut on a Reichert Ultracut-S microtome, picked up on to copper grids stained with lead citrate and examined in a JEOL 1200EX transmission electron microscope or a TecnaiG2 Spirit BioTWIN. Images were recorded with an AMT 2k CCD camera.