2.7 Immunohistochemistry SP Method

2.7 Immunohistochemistry SP Method

The remaining slices are subjected to immunohistochemical staining: O1. Follow the same steps as HE staining—first remove the wax from the slices; O2. Wash with PBS 2–3 times, 5 minutes each; O3. Add 3% H₂O₂ for 10 minutes to eliminate endogenous peroxidase activity and make the staining specific; O4. Wash with PBS 2–3 times, 5 minutes each; O5. Place the slices in a water bath containing 0.01 M sodium citrate buffer solution (pH 6.0) and heat to about 92–95°C for 15 minutes, allowing them to cool naturally to fully expose the antigen epitopes; O6. Repeat step 2; O7. At room temperature, add goat serum working solution for 10 minutes; O8. Add the primary antibody and incubate in a 37°C incubator for 2 hours; O9. After taking out the slices, wash them as before; O10. Add the biotinylated secondary antibody working solution and incubate in a 37°C incubator for 15 minutes; O11. After taking out the slices, wash them as before; O12. Add the horseradish peroxidase-labeled streptavidin working solution and incubate in a 37°C incubator for 15 minutes; O13. After taking out the slices, wash them as before; O14. Add reagents from the DAB chromogen kit to the slices, observe the coloration under the microscope, control the reaction time between 5 and 30 minutes, then wash; O15. Re-stain and then dehydrate, mount the slides; O16. Observe the slides under the microscope.