Protocol for Immunohistochemistry (Paraffin)
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.