Today's Weapon-Paraffin section immunohistochemical staining experiment operation flow
Paraffin section immunohistochemical staining experiment operation flow
1. Deparaffinize the paraffin sections to water: (The paraffin sections should be placed at 60 ° C for 1 hour before staining).
â‘ Xylene and II, 10 minutes each.
â‘¡ Gradient alcohol: 100%, 95% in 2 minutes, 80% in 2 minutes, 70% in 2 minutes and 2 minutes.
â‘¢ Washing with distilled water: 5 minutes, 2 times (put in a shaker).
2. Hydrogen peroxide to block endogenous peroxidase: 3% H2O2, room temperature for 10 minutes (protected from light).
3. Washing with distilled water: 5 minutes, 2 times (put in a shaker).
4. Antigen retrieval: select the appropriate method according to the antigen to be tested.
Attachment: Preparation of antigen recovery solution (10 mM pH 6.0 sodium citrate buffer): â‘ Preparation of stock solution: A solution: trisodium citrate-2H2O 29.41g + distilled water 1000ml; B solution: citrate 21g + distilled water 1000ml ; â‘¡ Preparation of working solution: A solution 82ml + B solution 18ml + distilled water 900ml
Antigen recovery method: ①Pressure cooker treatment technology: sodium citrate buffer (10mM, PH6.0), submerge the slices, cover the lid, boil in the pressure cooker, and slowly cool after 3 minutes of steam (you can use tap water to wash outside the pressure cooker, To help cool). ② Microwave processing technology: use a plastic slicing rack, place it in a plastic or temperature-resistant glass container, submerge the slices with sodium citrate buffer, choose medium high or high grade, 5 minutes; remove and replenish the preheated sodium citrate buffer ; Choose medium high or high grade again, 5 minutes (. The optimal temperature is 92 95 ℃) ③ Enzyme digestion treatment.
Precautions for antigen repair: â‘ The tissue cannot be dried. â‘¡ The method of antigen retrieval depends on the antibody. â‘¢ This method is mainly used for 10% formalin fixation and paraffin-embedded tissue. â‘£ PBS buffer is required for the process from antigen recovery to DAB color development.
5. PBS: 5 minutes, 2 times (put on a shaker).
6. Normal serum blocking: remove the slice from the staining cylinder, wipe off the moisture on the back of the slice and the moisture around the tissue on the front of the slice (to keep the tissue in a moist state), and add normal goat or rabbit serum (animal serum homologous to the second antibody) ) Treatment, 37 ° C, 15 minutes.
Attachment: normal serum preparation (or according to the concentration specified in the kit): 1:20 ratio, prepared with PBS, the required amount of each slice is calculated by 50μ + 5μl (10% throwing amount).
7. Dropping the first antibody: Absorb the serum with filter paper, without washing, add the first antibody directly at 37 ℃ for 2 hours (it can also be placed in a refrigerator at 4 ℃ overnight).
8. PBS: 5 minutes, 2 times (put on a shaker).
9. Add the biotinylated secondary antibody dropwise at 37 ° C for 40 minutes.
10.PBS: 5 minutes, 2 times (put on a shaker).
11. Add the third antibody (SAB complex) dropwise at 37 ° C for 40 minutes.
12.PBS: 5 minutes, 2 times (put on a shaker).
13. DAB color development, observe under the microscope, and terminate in time (the tap water is terminated).
Attachment: Preparation of DAB ①Preparation of stock solution (DAB 25mg / ml): DAB 250mg + PBS 10ml, after complete dissolution into 1ml, 100μl, 50μl, 20μl, etc., -20 ℃, frozen. ② Working solution: DAB stock solution 20μl + PBS 1000μl + 3% H2O 2 5μl
14. Rinse thoroughly with tap water (fine water).
15. Hematoxylin counterstaining, room temperature, 30 seconds, rinse with tap water.
16. Rinse with tap water and return to blue for 15 minutes.
17. Gradient alcohol dehydration: 80%, 95% in 2 minutes, 100% in 2 minutes, 2 times, 5 minutes.
18. Xylene transparent: I, II (xylene) for 5 minutes each
19. Sealing: Canadian gum (or neutral gum) sealing.
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