Protocol for Immunohistochemistry (Paraffin)

Cloud-Clone Corp.

1. Baking:Bake the IHC slides for 0.5~2 hours at60℃.

2. Deparaffinization and rehydration: After thebaking, put slides into the following reagents in order:

    1)XyleneI: 30 minutes

    2)XyleneII: 30 minutes

    3)100%ethanol: 10 minutes

    4)95%ethanol: 10 minutes

    5)80%ethanol: 10 minutes

    6)70%ethanol: 10 minutes

    7)Rinse sections with running water 10 minutes

3. Antigen retrieval - Microwave heat antigenretrieval (optional): Transfer the slides into a boiling EDTA-TRIS solution,and ensure the slides are soaked completely. Low fire for one minute, stopheating and keep in microwave for 10min, then another low fire for 3-4 min, andallow slides to cool in the EDTA-TRIS solution at room temperature.

4. Rinse 3X in PBS: Remove the residual, soak in PBS, and rinse in shakerat 40 RPM, 5min each time.      

5. Blockingendogenous peroxidase: Incubate the slides with 3%-5% H2O2.Solution for 10min at room temperature.

6. Rinse3X in PBS: Remove the residual, soak in PBS, and rinse in shaker at 40 RPM,5min each time.

7. Blocking: Remove the residual, incubate theslide with 5% BSA solution for 20min at room temperature.

8. Incubation primary antibody: Remove theresidual, incubate the slides with diluted primary antibodyovernight at 4℃ (recommended) or for 1 hour at 37℃. 

9. Rewarming of slides: Move the slides from 4oCto room temperature for about 20min to avoid the off-slide for rinseimmediately.  

10. Rinse3X in PBST: Remove the residual, soak in PBST, and rinse in shaker at 0 RAM,5min each time.                                                            

11. Incubation HRP-conjugated secondary antibody:Remove the residual, incubate the slides with diluted HRP-conjugated secondaryantibody at a appropriate dilution for 60min at 4℃.

12. Rinse3X in PBST: Remove the residual, soak in PBST, and rinse in shaker at 40RAM, 5min each time.

13. Chromogenic detection: Usea DAB chromogenic kit (Cat # IS046) to stain the tissue section. Mix Reagent Aand B thoroughly as the manual, then add appropriate volume to the tissuesection and incubate at room temperature for 0.5-10 minutes. End the reactionby washing the tissue section with distilled water till a brown color develops.

14. Slightly counterstain the tissue sectionwith haematoxylin.Then dry the tissue section, and put on a drop of resin sealthe tissue section with a cover slip. The tissue section is ready forobservation under a microscope.