【IHC experiment】Preparation of paraffin-embedded tissue sections.
Paraffin-embedded tissue section preparation is an essential step in immunohistochemistry (IHC) experiments, the purpose of which is to fix and embed tissue samples with paraffin wax to make them suitable for sectioning and immunohistochemical staining. The following are the detailed steps and precautions for the preparation of paraffin-embedded tissue sections.
1. Material collection and fixation.
When collecting materials, representative tissue samples should be selected as much as possible and quickly placed in pre-cooled 4% paraformaldehyde for fixation to maintain the morphology and structure of the cells. The fixation time is generally 30 minutes to 1 hour, depending on the type and size of the tissue.
2. Dehydration and transparency.
Dehydration is an important step in the preparation of paraffin-embedded tissue sections, the purpose of which is to remove water from the tissue in preparation for the next step of wax immersion. The commonly used dehydrating agent is gradient alcohol, which is gradually replaced from low concentration to high concentration, and needs to stay for a few minutes to ten minutes each time it is replaced to ensure the dehydration effect. Xylene is generally used as a clarifier, which can quickly remove the alcohol in the tissue and make the tissue transparent.
3. Wax immersion and embedding.
Wax immersion is the soaking of tissue in hot paraffin wax to allow paraffin to penetrate into the tissue and replace the clearing agent in the tissue. This step requires a good grasp of the temperature and time to prevent the tissue from overheating or paraffin solidification. Embedding is to put the wax-impregnated tissue into a mold and then pour hot paraffin wax to seal it. In order to make the slices easy to fall off, the tissue should be placed flat at the bottom of the mold during embedding, and the tissue should be compacted with a tool such as a small stick.
4. Slicing and patching.
The embedded tissue block is sectioned, usually 3-5 microns thick. Cut sections should be immediately attached to the slide and allowed to dry naturally at room temperature. In order for the sections to adhere better to the slides, protein glycerol or other adhesives can be added dropwise to the surface of the slides.
5. Precautions.
1.When collecting and fixing, a representative tissue sample should be selected and quickly fixed to preserve the morphology and structure of the cells.
2.When dehydrating, it is necessary to grasp the replacement sequence and residence time of gradient alcohol to ensure the dehydration effect.
3.When impregnation and embedding, the temperature and time should be controlled to prevent the tissue from overheating or paraffin solidification.
4.When slicing, the thickness and uniformity should be controlled to ensure the accuracy and comparability of staining results.
5.When patching, choose the appropriate adhesive to ensure that the section is flat and firmly attached to the slide.
6.Pay attention to safety during operation and avoid contact with toxic chemicals and high-temperature heated items.
7.Maintain cleanliness and hygiene in the laboratory to prevent cross-contamination and bacterial contamination.
8.After the experiment, the instruments and utensils should be cleaned in time, and the waste should be disposed of in accordance with the laboratory regulations.
In conclusion, paraffin-embedded tissue section preparation is one of the important steps in IHC experiments, and every detail needs to be taken seriously to ensure the accuracy and reliability of the experimental results. Through continuous practice and exploration, we can further improve the technical level of paraffin-embedded tissue section preparation and provide better services for scientific research and clinical diagnosis.