Frozen Tissue Sectioning Protocol
GUIDELINE
Frozen tissue sectioning is the use of freezing to harden the tissue in a relatively short period of time, which can be cut into very thin sections and applied to various examinations. While frozen sections are physically less stable than paraffin, they are especially superior in the preservation of antigenicity and lipid retention. It is the most widely used in histological techniques, especially for rapid intraoperative pathological diagnosis of clinical surgery patients and histochemical and immunohistochemical diagnostic studies in scientific research.
METHODS
- Fresh material should be taken as quickly as possible to prevent post-mortem changes in the tissue.
- Place the tissue block flat in a soft plastic bottle cap or special box (about 2 cm in diameter). If the tissue block is small, add OCT embedding agent in appropriate amount to submerge the tissue, and put the special cassette into the cup with liquid nitrogen slowly and flatly. When the bottom of the box is in contact with the liquid nitrogen, it starts to boil, at this time the box remains in place, do not dip into the liquid nitrogen, about 10-20 s tissue is rapidly frozen into a block.
- The sample tray is coated with a layer of OCT embedding gel, and the quick-frozen tissue is placed on it, and the 4°C refrigerator was pre-chilled for 5-10 min to let the OCT gel soak through the tissue. Remove the tissue and place it on a tin foil or slide, and snap freeze the sample tray. The tissue is placed on the sample tray with another layer of OCT adhesive on it to completely cover the tissue, and snap frozen on the rack (PE) for 30 min.
- The basic structure is that the slicer is placed in a low-temperature sealed room, so the slicing is not affected by the external temperature and environment, and can be continuously sliced to 5-10μm. When slicing, the temperature in the low-temperature room is -15°C to -20°C is appropriate, the temperature is too low and the tissue is easy to break, the position and angle of the anti-roll plate should be appropriate, and the slide should be attached to the tissue section, do not move up and down.
- After cut and placed at room temperature for 30 min, fixed in acetone at 4°C for 5-10 min and oven dried for 20 min. 5 min x 3 for PBS wash.
NOTES
- After the block is frozen, it can be placed in a constant-cooling chamber for freezing and slicing. If it needs to be stored, it should be quickly sealed with aluminum foil or plastic film and immediately placed in -80°C refrigerator for storage.
- The key to making frozen tissue sections lies in the adjustment of the anti-collapse plate, which requires the operator to be careful and accurately adjust it to the proper position.
- When the cut tissue is adhered to a clean slide, a slight force in one direction can avoid folding of the tissue during the spreading process and ensure the integrity of the tissue structure and the beauty of the slice.
