This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Preparation of paraffin-embedded tissue samples

Materials and Instruments

Tissue. Trypsin.
Xylene Ethanol Saline
Nylon mesh

Move

1. Cut the paraffin-embedded tissue into 3-5 pieces of 40-50 μm thickness, or use a milk bowl to grind into 0.5 mm diameter granules, and put them into a 10 ml test tube.

2. Add 5~8 ml of xylene and dewax at room temperature for 1~2 d. Depending on whether the paraffin wax is clean or not, replace the xylene 1~2 times.

3. Hydration: Add 5%, 95%, 70%, 50% gradient ethanol, each step is 10 min, remove ethanol, add 3-5 ml of distilled water, and discard after 10 min.

4. Digestion: add 2 ml of 0.5% trypsin (pH 1.5~2.0) digestion solution, and put it in a 37℃ constant temperature water bath for 30 min, and during the digestion period, use an oscillator to oscillate once every 10 min.

5. After 30 min of digestion, add saline immediately to terminate digestion.

6. Filter through 300 mesh nylon mesh, and the undigested tissue can be digested for the second time.

7. Collect the cell suspension, centrifuge and precipitate at 1500 r/min, rinse with saline for 1~2 times, centrifuge and precipitate at 500~800 r/min, and remove the debris.

8. Save the cells for later use.

Caveat

1. Be sure to remove the paraffin wax from the tissue, generally the first time should be about 12 h, the second time is about 30 min. To test whether the paraffin wax has been removed, discard xylene and add anhydrous ethanol, if no flocs float up, the wax can be regarded as having been removed; otherwise, the wax has not been removed.

2. the digestion time should not be too long, so as not to cause the released cell nuclei to be digested. 3. the slices should not be too thin or too long.

3. the slice should not be too thin or too thick, too thin cell debris is too much, affecting the analysis results; too thick cause undesirable dewaxing.

4. The digested tissue should be cut with ophthalmic scissors first, then digested with pepsin for 30 min at 37℃, and then blown into suspension with a pointed pipette.

5. The digested tissue suspension may have a small number of cells after filtration, so that the tissue pieces should be placed on a 300-mesh nylon mesh, ground with a rounded test tube at the bottom, and then rinsed with PBS liquid; so repeated, you can get a larger number of single-cell suspension.

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