Multiple immunohistochemical detection

Multiple immunohistochemical detectionExperimental procedure:

1. Cut the embedded specimen into 3um sections using a paraffin slicer

2. Bake the slices at 60 ℃ for 1 hour

3. Xylene I dewaxing for 10 minutes

4. Xylene II dewaxing for 10 minutes

5. Gradient alcohol rehydration: 100% alcohol for 5 minutes, 100% alcohol for 5 minutes, 95% alcohol for 5 minutes, 80% alcohol for 5 minutes, rinse with tap water for 5 minutes

6. Antigen repair: Place the slices in a 0.1mol/L citric acid solution with a pH of 6.0 for 20 minutes in a boiling water bath. Stop heating and cool naturally for 20-30 minutes

7. Use PBS for 3 minutes and 3 times, draw circles in groups, add hydrogen peroxide dropwise, and incubate for 20 minutes

8. Use PBS 3 times every 3 minutes, and add blocking serum dropwise to block for 1 hour

9. Shake dry, add a suitable concentration of primary antibody dropwise, and incubate overnight at 4 ℃ in a wet box

10. Wash the primary antibody with PBS 3 minutes x 5 times

11. Shake dry, add secondary antibody dropwise and incubate in an incubator at room temperature for 1 hour (note the species of secondary antibody, 30 minutes in summer and 1 hour in winter)

12. Wash the secondary antibody with PBS 3 minutes x 5 times

13. Shake dry, add DAB dropwise for color development, rinse with water

14. Stain the nucleus with hematoxylin for 5 minutes (depending on the new configuration of hematoxylin)

15. Rinse excess hematoxylin with running water and differentiate for 1 second in the differentiation solution

16. Rinse with running water for 5 minutes

17. Gradient alcohol rehydration: 80% alcohol for 5 minutes, 95% alcohol for 5 minutes, 100% alcohol for 5 minutes, 100% alcohol for 5 minutes, xylene II transparent for 5 minutes, xylene I transparent for 5 minutes (if fully transparent, the time can be appropriately extended)

18. Seal with neutral gum

19. Microscopic examination