To maintain ultrastructure, specimens must be fixed rapidly after the oxygen supply has been cut off. The specifics of fixation (fixative, concentration, time, temperature) and the buffering medium will vary for specific experiments. However, in general, fixation will involve buffered aldehydes which cross link proteins, carbohydrates, and to a certain extent lipids. The fixatives will cross link similar molecules that surround the sample (body fluids, cell culture medium, etc. ) so the sample should be washed briefly in a buffered solution before fixation.
Aldehyde fixatives penetrate tissue slowly, so samples must be small (less than 0.5 mm on two sides). For large specimens, mincing the sample directly in fixative after washing works best. This can be done by placing a drop of fixative on dental wax or parafilm and placing the excised and washed sample in the drop of fixative and then mincing with fresh razor blades. The EM staff can show you how this is done.
Cold depolymerizes cytoskeletal elements, so whenever possible, buffer washes, fixatives, and sample should be at room temperature or warmer to initiate fixation. For temperature sensitive samples, such as enzyme cytochemistry, the samples can be briefly warmed placed in fixative and then, after a minute, re-cooled. To store fixed samples for more than a few hours, the specimen should be placed on ice or in the refrigerator after fixation. For storage of samples more than a few days before processing, the fixative should be replaced with buffer after 24 hours.Submitting Samples
All samples must now go through the EM online laboratory information management system located here. Please contact Janice, Mary, or Maria, if you experience any problems or need access to the system.
Please note that specimens for EM processing should be fixed prior to submission and kept refrigerated until submitted. EM fixative and wash buffer can be obtained from the refrigerator in the Processing Lab located at T-3208 MCN.