Sending organelles on glass for deep-etch EM
Our standard protocol is to adsorb dilute suspensions of membrane fractions & cell organelles onto tiny 2mm x 2mm glass coverslips that have been chromate-cleaned, washed extensively in water, and coated with 1mg/ml of high MW polylysine (300-400kDa). Typically, we place 50ul of a slightly turbid suspension onto each coverslip and leave it there for 1-3 min, then rinse the glass in whatever solution the membranes were originally suspended in, with 2% glutaraldehyde added to fix the membranes in place on the polylysine.

Next, we wash the coverslips extensively in distilled water to remove all the glutaraldehyde and all the salts, & sugars that the membranes were in. (The reason for this water-wash -- the most problematic step in the whole procedure because it exposes the membranes to a highly unnatural environment -- is that ANY residual salt or sugar will leave an unacceptable “scum” on the coverslips after freeze drying.)

Next, we carefully blot excess water of the coverslips without allowing them to air dry completely, and then quick-freeze them by impacting them against a pure copper block cooled to minus 269 degrees Celsius with liquid helium. We then freeze-dry the coverslips at minus 80 degrees Celsius for 15 min, “replicate” them with a thin ~2nm coating of platinum, float the replicas off the glass by angled immersion into concentrated hydrofluoric acid, wash the replicas with water and clean them by floatation on household bleach and further washes, pick them up on 75 mesh formvar-coated grids, and view them in the transmission electron microscope.

We can’t tell you how much material to send us the first time. We need a total of at least 100ul of each type of suspension for each variable you might want to try out, but how readily your particular membrane fractions will adsorb to the glass is at first unknown. If you could try for the first time send us HALF of one of your usual preps the first time, keeping the other half for your own biochemical analysis, that would seem appropriate.

Please make sure to send your samples to the specific address indicated in my signature, below. Put them in a Styrofoam box in crushed ice. (DO NOT INSERT “picnic ice” or other sorts of ethylene glycol packs in the shipment, as these tend to freeze samples during transport.) Of course, pack the ice in a ziplock bag and seal the Styrofoam box securely with tape so that it won’t leak during shipment, as this irritates FedEx considerably. In addition, don’t forget to send your samples FedEx PRIORITY. The FedEx “standard” next-day shipment generally comes in too late in the day for us to have time to work the deliveries up the same day.