Esophageal Adenocarcinoma Methods and Protocols

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3. Put the chosen and labeled cassette on top of the paraffin in
   the mold.


4. Then, put a small amount of melted paraffin in the mold to
   pack cassette with paraffin while cooling, keeping the mold full
   until solid. The paraffin should solidify in 30 min.


5. When the wax is fully cooled and hardened, transfer it to a
   metal cold plate.


6. Cut all irregularities from the block holders and check that the
   paraffin is clear of any holes caused by the generation of air
   bubbles during block preparation. For backup, add hot paraf-
   fin to the face of the plastic cassette and be sure there is enough
   paraffin to cover the face of the plastic cassette. The paraffin
   block can be easily popped out of the mold. The wax blocks
   should not stick to the mold. If the wax cracks, simply melt it
   again and start over. It is important that the “recipient” block
   should not have any crack.

“Donor” paraffin blocks are blocks that donate small portion of

tissues into the “recipient” paraffin block (TMA).

1. Collect the pathology report as well as the histological slides of
   the samples.


2. Review the histology of the samples to choose the block of
   interest in each patient for the TMA.


3. Representative areas of each slide must be marked on the slide
   to guide the person who will do the punching work.


4. Retrieve the corresponding paraffin blocks of all the chosen
   cases. Importantly, each “donor” block must be at least 1 mm
   thick to be suitable for constructing tissue arrays. However, for
   better results, we suggest the “donor” tissue blocks with
   3–4 mm thickness. A factor that affects tissue loss rates in TMA
   is the quality of the tissue. If “donor” blocks are too dry, they
   are at higher risk of breaking into fragments or detaching from
   “recipient” block.


5. For keeping the information in the right order, it is important
   to generate an Excel database. The database should include the
   specimen identifier in a way that the specimens are aligned in
   the targeted TMA block. Some machine may have software for
   this purpose.

The adjustment of the specimens on the array is important because

confusion about their localization can threaten the evaluation of

the experiment. We need to use a few normal tissues, from “donor”

blocks, in each TMA as control tissues. These normal tissues have

two major purposes. The first is to add as positive controls for

some expression experiments. The second purpose is to put them

2.2 “Donor” Paraffin
Blocks

2.3 Control Tissue
Blocks

TMA and Esophageal Adenocarcinoma