Tissue Embedding for Photography

In order to obtain a clear view of the hippocampal tissue slice in the Epon block for light microscopy photography, the following procedure was developed. This procedure includes placing the tissue slice to the bottom of the flat mold and, later in the curing process, attaching some Aclar to one side of the hardened block.

Materials: hippocampal tissue slice, flat coffin mold, Epon resin, wooden applicator stick, plastic transfer pipet, Aclar plastic film (Ted Pella, Inc., Redding, CA), 100 mm diameter plastic Petri dish, pair of scissors or a single edge razor blade, 60 degree C oven, stereomicroscope.

Procedure:

A. Embedding the tissue

1. Place the tissue’s paper label onto the floor of the flat coffin mold.

2.  Fill the mold carefully with the Epon resin mixture, using the plastic transfer pipet. Avoid introducing any air bubbles. Fill the mold to about 1/3 full with Epon resin.

3. Squeeze out any air bubbles trapped under the paper label, using the wooden applicator stick.

4. Allow this resin to partially polymerize in a 60 degree C oven for about 2 and ½ hours. Then remove the mold/resin from the oven.

5. The resin should be of a gooey consistency but still allow easy placement of the hippocampal slice to the floor of the mold. Use the wooden applicator stick to position the tissue slice on the bottom of the mold. Remove any surrounding air bubbles. Use a stereomicroscope to observe your efforts.

6. Next, place the mold into the 60 degree C oven for 24 to 36 hours (depending on when is most convenient time for you to do the Aclar step).

B. Attaching Aclar to the block

1. Remove the mold from the oven to cool to room temperature. Place it on a counter-top in the fume hood.

2. Once it has cooled, remove the block from the mold.

3. Cut a small square of Aclar, about an inch or two square. Place this square of Aclar onto the floor of the plastic Petri dish.

4. Add a drop or two of fresh Epon resin onto the center of the square of Aclar.

5. Place the block, tissue side down on top of the Epon drop, without creating any air bubbles.

6. Place the Petri dish into the 60 degree C oven for additional hours of curing. If the block has already been cured for 24 hours, then leave it in for another 24 hours. If the block has been cured for 36 hours, then leave it for another 12 hours. In all, the block should receive a total of 48 hours of 60 degree C heat.

7. When the block is due to come out of the oven, remove it and allow it to cool to room temperature in the fume hood.

8. Next, carefully peel away the Aclar plastic film. The Aclar side of the block should now be very smooth and transparent. This side is the photographable side. Place the block into a protective container for storage.