【IHC experiment】Preparation of paraffin-embedded tissue sections.
First, the purpose of the experiment.
The purpose of this experiment was to learn and master the preparation process of paraffin-embedded tissue sections for subsequent immunohistochemical staining (IHC) experiments. Through this experiment, we will understand the importance of section preparation of paraffin-embedded tissues for subsequent research, and learn to operate related equipment to lay the foundation for subsequent experiments.
Second, the principle of experiment.
Paraffin-embedded tissue section preparation is to use paraffin wax to embed the tissue, and after a series of treatments and sections, the sections can be prepared for subsequent experiments. In this experiment, paraffin will be used to fix and preserve the tissue, and the tissue will be cut into thin sections by a microtome for subsequent staining and observation.
3. Experimental procedures.
1.Tissue collection: Select appropriate tissue blocks, rinse them with normal saline, and blot them dry with filter paper.
2.Fixation: Place the tissue block in formaldehyde buffer and fix for 24 h.
3.Water washing: Rinse the fixed tissue block with water for 10 minutes and change the water three times.
4.Dehydration: Tissue blocks are sequentially placed in % and 100% alcohol for 1 h per gradient.
5.Paraffin embedding: The dehydrated tissue block is placed in paraffin and embedded.
6.Sectioning: Slice paraffin-embedded tissue into 5-7 micron slices using a microtome.
7.Patch: Attach the slices to the slide and tumble dry.
8.Deparaffinization: Put the slide after the patch into xylene for 10 minutes and change the xylene once.
9.Staining: H&E staining or other staining methods as needed.
10.Mounting: Cover the stained slides with neutral gum.
4. Precautions.
1.During the dehydration process, it is necessary to ensure that the time of each gradient is accurate to avoid affecting the results of subsequent experiments.
2.The temperature should not be too high during embedding, so as not to damage the tissue structure.
3.When slicing, adjust the thickness of the slice to ensure the integrity and consistency of the slice.
4.During the deparaffinization and staining process, it is important to ensure that the slide does not move so as not to affect the experimental results.
5.Neutral gum should be filtered before use so as not to affect the mounting effect.