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Tissue embedding is to embed tissue specimens in paraffin after fixation, sampling, and dehydration.
I believe most laboratory managers will emphasize the importance of organizational processing to employees. Embedding is very important for preserving tissue morphology and providing tissue support during sectioning.
1. Materials: fresh tissues were fixed in 4% paraformaldehyde(PFA) for at least 24 hours. Take out the tissue from the fixed solution, trim the target tissue with a Sterile Scalpel in the fume hood, and place the trimmed tissue and the corresponding label in the dehydration box.
2. Dehydration: put the dehydration box into the hanging basket and dehydrate by the gradient alcohol in order.
75% Alcohol 4h,85% Alcohol 2h,90% Alcohol 2h,95% Alcohol 1h
Absolute Ethanol I 30min, Absolute Ethanol II 30min
Alcohol Benzene 5-10min
Xylene I 5-10min, Xylene II 5-10min
Wax I 1h, Wax II 1h, Wax III 1h
To learn more about the dehydration steps in tissue processing, click to Download the guidance document.
3. Embedding: Embedding wax-soaked tissue in the embedding machine. First, put the melted wax into the embedding frame, take out the tissue from the dehydration box before the wax solidified.
Secondly, put it into the embedding frame according to the requirements of the embedding surface, and affix the corresponding label. Cool on the - 20 °freezer, remove the wax block from the embedding frame and trim after the wax solidifies.
4. Slicing: Place the trimmed wax block on a paraffin microtome, with a thickness of 4μm. The slices were floated in 40℃ warm water of the spreader to flatten the tissue. The tissue was picked up with a Glass Slide and put into a 60℃ oven to bake the slices. After the water is dried and the wax is roasted, take it out and store it at room temperature for later use.
1. Ensure that the fixative volume is 10 to 15 times the tissue block.
2. Choose different fixatives according to tissues and experimental purposes.
3. Some fixatives have a strong hardening effect on the tissue, and the action time should be strictly controlled and should not be too long.
4. The section of the material shall be flat, and the thickness shall not exceed 2mm, otherwise the dehydration effect cannot be guaranteed.
5. For the tissues with licensing requirements, the complete retention of the target tissues shall be ensured when taking materials.