Use IHC/ISH Peroxidase Block to block nonspecific background staining observed during immunostaining.
Product Details
| Applications: | IHC
ISH (in situ hybridization)
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| Shipping: | Blue Ice Not Frozen |
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| Long Term Storage: | +4°C |
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| 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. |
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| Protocol: | Recommended protocol for IHC (Immunohistochemistry)
- Arrange formalin-fixed paraffin-embedded (FFPE) tissue sections in a slide holder.
- Heat in a drying oven at 55-60°C for 20 minutes.
- Deparaffinize with a Xylene solution (or Xylene substitute) for 10 minutes.
- Follow with 100% ethanol for 6 minutes.
- Rehydrate slides for 1 minute in each step: 90%, 70%, 50% ethanol and finally in distilled or deionized water.
- 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.
- Wash for 5 minutes with PBST (Phosphate Buffered Saline Tween-20).
- Wipe excess liquid around the section on the glass slide and encircle the tissue section with a PAP pen.
- Dry slide for 10 seconds
- Incubate the tissue with IHC/ISH Peroxidase Block for 5 minutes.
- Wash with PBST for 2 minutes.
- Researcher needs to optimize the concentrations and incubation times for biotinylated primary antibodies (recommended time: 20 to 30 minutes)
- Wash with PBST for 5 minutes.
- 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.
- Wash with PBST for 5 minutes.
- Incubate sections with appropriate Blocker/Diluent to cover the tissue section for 10 minutes to block non-specific binding of detection reagent.
- Use detection reagents and HIGHDEF® Chromogen to visualize antigens.
Recommended Protocol for ISH (In Situ Hybridization)
- Arrange formalin-fixed paraffin-embedded (FFPE) tissue sections in a slide holder.
- Heat in a drying oven at 55-60°C for 20 minutes.
- Deparaffinize with Xylene (or Xylene substitute) for 10 minutes.
- Follow with 100% ethanol for 6 minutes.
- Rehydrate slides for 1 minute in each step: 90%, 70%, 50% ethanol and finally in distilled or deionized water.
- Wipe excess liquid around the section on the glass slide and encircle the tissue section with a PAP pen.
- Dry slide for 10 seconds.
- Expose the tissue to IHC/ISH Peroxidase Block for 5 minutes.
- Wash with washing buffer for 2 minutes.
- Place the slide on a 37°C warm plate.
- 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.
- Wash with PBST for 5 minutes.
- 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).
- Add denatured probe to the section.
- Cover with cover slip.
- Perform stringent wash after hybridization, with 40% Formamide + 6X SSPE buffer for 10 minutes.
- Wash with PBST (Phosphate Buffered Saline Tween-20) for 5 minutes.
- Incubate sections with appropriate Blocker/Diluent to cover the tissue section for 10 minutes to block non-specific binding of detection reagent.
- Use detection reagents and HIGHDEF® Chromogen to visualize antigens.
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| Regulatory Status: | RUO - Research Use Only |
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Product Literature References
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15, 1164 (2023),
Abstract;
T-cell mediated targeted delivery of anti-PD-L1 nanobody overcomes poor antibody penetration and improves PD-L1 blocking at the tumor site: P.F. Petit, et al.; Cancer Immunol. Res.
1158, 2326 (2022),
Abstract;
Caspase recruitment domain (CARD) family (CARD9, CARD10, CARD11, CARD14 and CARD15) are increased during active inflammation in patients with inflammatory bowel disease: J.K. Yamamoto-Furusho, et al.; J. Inflamm. (Lond.)
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