In Situ Embedding in Epoxy Resin of Tissue Cultured in Leighton Tubes; Selection of Single Cells for Electron Microscopy

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Abstract
Processing is carried out in the original culture vessel as follows: fixation in a 1.0% solution of phosphate-buffered OsO4 at 5 C, 30 min; dehydration in ethanol, 30 min; propylene oxide, 30 min; infiltration with a 1:1 mixture of resin and propylene oxide, 30 min; and undiluted resin at 5 C, 12 hr. This resin is replaced with 8 ml of fresh, and the tube is placed horizontally in an oven. Araldite is polymerized at 55 C for 21 hr; Maraglas, 60 C for 24–48 hr. Immediately upon removal from the oven, the hot tube is plunged into an ice-water bath at 0–0.5 C. The tube usually cracks extensively, and after 8–10 min soaking, the glass fragments can be easily removed. If a tube fails to break, it can be cracked mechanically, then allowed to soak until separation occurs. Advantages of the method are that an entire culture can be embedded, the resin cast separates cleanly, and any part of the culture becomes available for light and electron microscopy.