IHC/ISH Peroxidase Block

Manuals And Inserts

• Datasheet

Specific Protocol

Recommended protocol for IHC (Immunohistochemistry)

1. Arrange formalin-fixed paraffin-embedded (FFPE) tissue sections in a slide holder.
2. Heat in a drying oven at 55-60°C for 20 minutes.
3. Deparaffinize with a Xylene solution (or Xylene substitute) for 10 minutes.
4. Follow with 100% ethanol for 6 minutes.
5. Rehydrate slides for 1 minute in each step: 90%, 70%, 50% ethanol and finally in distilled or deionized water.
6. Retrieve Antigens with Antigen Retrieval Reagent (Citrate, pH 6.0) or Antigen Retrieval Reagent (Tris-EDTA, pH 9.0) for 20 minutes at 99°C.
7. Wash for 5 minutes with PBST (Phosphate Buffered Saline Tween-20).
8. Wipe excess liquid around the section on the glass slide and encircle the tissue section with a PAP pen.
9. Dry slide for 10 seconds
10. Incubate the tissue with IHC/ISH Peroxidase Block for 5 minutes.
11. Wash with PBST for 2 minutes.
12. Researcher needs to optimize the concentrations and incubation times for biotinylated primary antibodies (recommended time: 20 to 30 minutes)
13. Wash with PBST for 5 minutes.
14. If unlabeled mouse primary antibodies are used, sections must be incubated with biotinylated anti-mouse antibody and if unlabeled rabbit primary antibodies are used, use biotinylated rabbit anti-mouse linker (diluted in blocker/diluent) for 15 minutes.
15. Wash with PBST for 5 minutes.
16. Incubate sections with appropriate Blocker/Diluent to cover the tissue section for 10 minutes to block non-specific binding of detection reagent.
17. Use detection reagents and HIGHDEF® Chromogen to visualize antigens.

Recommended Protocol for ISH (In Situ Hybridization)

1. Arrange formalin-fixed paraffin-embedded (FFPE) tissue sections in a slide holder.
2. Heat in a drying oven at 55-60°C for 20 minutes.
3. Deparaffinize with Xylene (or Xylene substitute) for 10 minutes.
4. Follow with 100% ethanol for 6 minutes.
5. Rehydrate slides for 1 minute in each step: 90%, 70%, 50% ethanol and finally in distilled or deionized water.
6. Wipe excess liquid around the section on the glass slide and encircle the tissue section with a PAP pen.
7. Dry slide for 10 seconds.
8. Expose the tissue to IHC/ISH Peroxidase Block for 5 minutes.
9. Wash with washing buffer for 2 minutes.
10. Place the slide on a 37°C warm plate.
11. Add the optimal concentration of Proteinase K (Recommended: 25-80 μg/mL, varies with tissue type and thickness) and incubate for 10 minutes at 37°C.
12. Wash with PBST for 5 minutes.
13. Researcher needs to optimize the concentrations and incubation times for biotinylated RNA or DNA probes (recommended: 10 minutes of denaturation at 95°C and at least 120 minutes for hybridization, typically at 37°C for DNA probes, and 42°C for RNA probes).
14. Add denatured probe to the section.
15. Cover with cover slip.
16. Perform stringent wash after hybridization, with 40% Formamide + 6X SSPE buffer for 10 minutes.
17. Wash with PBST (Phosphate Buffered Saline Tween-20) for 5 minutes.
18. Incubate sections with appropriate Blocker/Diluent to cover the tissue section for 10 minutes to block non-specific binding of detection reagent.
19. Use detection reagents and HIGHDEF® Chromogen to visualize antigens.

Certificate of Analysis

Please enter the lot number as featured on the product label

Lot No. :

SDS

Enzo Life Science provides GHS Compliant SDS

• English (EU) SDS

If your language is not available please fill out the SDS request form