Embedding is to take the fixed, dehydrated, transparent, and wax-impregnated tissue blocks from the final wax bath and place them into an embedding frame filled with molten paraffin, and then embed them into blocks so that the tissue and the embedding agent can fuse together and quickly cool, this procedure is called embedding. After the embedding agent solidifies, the hardness and toughness of the tissue are further strengthened to facilitate sectioning. This article will now introduce the preparation work before embedding and precautions during embedding.

Figure 1. Slices from an intact macaque hemisphere embedded by paraffin, stained with H&E. (Zhanmu O, et al.; 2020)

Preparation before embedding:

• Instrument status check: check the power-on status and wax discharge status of the wax nozzle
• Preparation of embedding tools: tweezers, trays, garbage trays
• Preparations for embedding and contamination prevention: paper towels and cotton swabs.

Preparation before embedding: Checking the embedding handover order

• Prevent loss of wax blocks: check the total number of wax blocks before embedding
• Prevent tissue block loss: check the number of tissue blocks when embedding
• Prevent investment surface errors: use appropriate embedding surface and tissue

Preparation before embedding: The tissue is always soaked in wax liquid

• Liquid volume: 2/3 height of dehydration box
• Function: Keep the tissue in a wax-immersed state to avoid vacuoles and infusibility of the tissue with the wax liquid.

Basic Standards of Embedding

1. Select the Matching Embedding Cassette Size
   There should be a 1mm gap around the embedded tissue block and the edge of the embedding cassette, which means that the tissue block and the embedding cassette are matched. Choose a small embedding cassette that can embed small pieces of tissue; choose a cassette that can embed medium-sized embedding cassette.
2. How to Place the Dehydration Box
   Lay it horizontally, with the end with the pathology number facing to the left. Place it in the center and not offset to the left or right.
3. Inject Wax
   Fill the entire cassette at once, but do not overflow to avoid tissue dispersion caused by secondary wax injection.
4. Positioning of Embedded Tissue
   Centered and tightly packed, with no obvious gaps visible to the naked eye.
5. The Embedding Level Should be Consistent
   The thickness of the wax block is consistent, ensuring that the repair can be done simultaneously at one time.
6. Organizational Arrangement
   Neatly, square arrangement is preferred; if the square cannot be arranged, the arrangement should be rectangular, and should be in vertical rows with the same arrangement direction.
7. The Embedding Surface is Correct
   
   • If there is a requirement for sectioning, the maximum section required for sectioning should be face down. For those without section requirements, the maximum section should be face down.
   • The skin side, intestinal wall intima side, and cyst wall intima side face left
   • The clear layers of the lumen, cyst wall, skin, etc. should be buried vertically so that the tissue structure of each layer can be seen under the microscope after staining the sections.
     For example: the skin must have epidermis and dermis or subcutaneous tissue, the intestinal wall and stomach wall must have mucosa, submucosa, muscle layer, serosal layer, and the kidney must have cortex and medulla, etc.
8. The embedded wax block has no air bubbles, no other tissue contamination, and no contamination from tips, paper, etc.;
9. The tissue and the wax block are seamlessly integrated. The tissue is fully melted during embedding and there should be no solidification. The embedding speed should be fast to prevent the molten wax from solidifying before the tissue is embedded;
10. It is appropriate for the embedding paraffin to be 2-4°C higher than the melting point of paraffin. Both are in a melted state. In this way, the structure of the condensed wax block can be integrated with the embedding paraffin; otherwise: if the temperature of the embedding paraffin is If it is too low, the paraffin will begin to condense during embedding, and it will be too late to operate, easily bringing in air bubbles. In this way, the density of the embedded wax block will often be uneven after condensation, and the tissue will be separated from the paraffin, making it difficult to cut complete sections. In these cases, it is necessary to re-embedded in wax.

Embedding: Only take out and open one box at a time

Key Points:

• There is always only one wax block to be wrapped on the embedding table.
• Be gentle when opening the box
• When opening the box, pay attention to whether any tissue pops out.

Significance:

• Avoid the risk of tissue popping out when opening multiple wax block boxes, causing confusion.
• It is convenient to clean the embedding table at any time and reduce the risk of contamination between tissues.
• Reduce the risk of slice quality due to coagulation of tissue blocks caused by temperature drop due to lack of time for embedding.
• Reduce the time wastage caused by determining the pathology number when checking the number of tissue blocks.

Embedding: Arrange tissue on hot stage

Key Points:

• Do a good job in planning, fixing, positioning and orienting the organization on the hot stage
• Move the tissue smoothly to the cooling stage for cooling

Significance:

• Reduce the unevenness of the tissue caused by too slow embedding speed, excessive trimming, tissue damage, and cracks between tissues during sectioning.
• When the wax liquid solidifies, there are bubbles in the tissue, which can easily cause cavities in the sections.
• Insufficient fusion of tissue and wax liquid results in easy cutting and collapse, causing tissue damage. Subsequent work will be suspended and reworked and repackaged.

Related Products

1. Zhanmu O, et al.; Paraffin-embedding for large volume bio-tissue. Sci Rep. 2020, 10(1):12639.

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