Fast embedding of monolayer cultures for electron microscopy
Dehydration should start at 50% ethanol and proceed to 100% ethanol in just a few minutes. (Samples should never see lower ethanol concentrations, and should never be rinsed in distilled water, because in both cases they tend to swell.  It’s bad enough that they are going to shrink in the higher alcohols.)

Plastic embedding of coverslips is done by dipping the coverslips sequentially through nine small beakers containing the following:

(The coverslip should be held with a forceps and ‘swished’ around in each beaker for no less than 30 seconds, but no longer than 60 seconds, for each)

  1. 100% Ethanol (really dry!)
  2. 100% Ethanol, again
  3. 100% Ethanol, again
  4. 100% propylene oxide
  5. 100% propylene oxide, again
  6. 50/50 (V/V) propylene oxide and epoxy resin*
  7. 50/50 (V/V) propylene oxide and epoxy resin, again
  8. 100% epoxy resin
  9. 100% epoxy resin, again

(*The full mixture of epoxy resin, including the accelerator, is made up fresh right before use, and is outgassed by repeated vacuum-application many times, until all the bubbles come out of it.  This we do with the vacuum-oven that will be used for the final epoxy polymerization, but with the heater turned off, so the epoxy doesn’t get heated and start to polymerize.  This the most tedious step of the whole procedure....You’ll see!))

The coverslips are then set in aluminum dishes filled with a ~1mm deep layer of epoxy resin and placed in the vacuum oven (now warmed up to 65degC), and evacuated two or three times, to draw off any residual propylene oxide.

Finally, the coverslips are inverted over  “Beem” capsules (or large gelatin pills), held perfectly upright somehow, and filled to the very top with the same fresh epoxy resin (trap no bubbles!), and placed in the heated 65degC vacuum-oven for polymerization. (Don’t apply any vacuum at this point, though, or bubbles might get trapped under the coverslip.)

After polymerization, the coverslips are “popped” off the Beem capsules by the usual technique: touching the glass to a copper or brass block partially immersed in LN2, and then pulling it off the epoxy.

NOTE ON THE OPTIMAL TYPE OF SUBSTRATE TO GROW CELLS ON, FOR THE ABOVE EMBEDDING PROCEDURE:

(Actually, we abhor this technique for separating glass coverslips from polymerized epoxy, and have switched completely to growing all our monolayer cultures on Thermanox® coverslips (25mm diameter circles.  These have adhesive properties almost identical to Falcon tissue culture dishes (much better than glass), and much more importantly, they peel off the “Beem” capsules without the slightest problem, and without all the LN2 fuss....so long as one removes the sample from the 65degC oven and peels the plastic coverslip off the capsule immediately, e.g., while it is still hot!!

If you don’t know about these Thermanox coverslips, you can read up on them at the following links:

http://www.nunc.de/page.aspx?ID=226

http://www.emsdiasum.com/microscopy/technical/datasheet/72274.aspx

The only problem with Thermanox coverslips is that even though they’re clear and transparent, they have very poor optical properties, so you cannot image cells on them by looking through them, but must use a water-immersion objective in the LM, so as to image cells on them from above.