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IHC/ISH Peroxidase Block

 
ENZ-ACC107-0100 100 ml Inquire for pricing
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Use IHC/ISH Peroxidase Block to block nonspecific background staining observed during immunostaining.

Product Specification

Applications:IHC
ISH (in situ hybridization)
 
Shipping:Blue Ice Not Frozen
 
Long Term Storage:+4°C
 
Technical Info/Product Notes:The IHC/ISH Peroxidase Block is a unique serum-free blocking solution used to reduce nonspecific background staining. It is a universal blocking agent which eliminates the need to match species with the link antibody. This blocking reagent has been shown to be superior to serum-based blocking solutions. In addition to immunohistochemistry, the IHC background blocker can also be used for ISH.

Interpretation of the results will be the sole responsibility of the user.
 
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.
 

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