(HRP-Polymer) Immunohistochemistry Kit (Rabbit) is a high-sensitivity immunohistochemistry kit. Horseradish peroxidase (HRP) is conjugated to the secondary antibody via polymer labeling to form large antibody-enzyme polymers, which replace the secondary and tertiary antibodies used in traditional methods. These polymers deliver potent signal amplification and excellent tissue and cell penetration, making them ideal for immunohistochemical applications. Compared with the conventional three-step method, this assay kit features simpler operation, faster testing, higher sensitivity and lower background staining.
Packing List:
| R777876 | Component | 1 Kit | Storage | | R777876A | Blocking Solution (5% BSA) | 6mL | 2-8℃ | | R777876B | Endogenous Peroxidase Blocker (3% H₂O₂) | 6mL | 2-8℃ | | R777876C | Anti-Rabbit IgG (HRP-Polymer) | 6mL | 2-8℃ | | R777876D | DAB Chromogenic Reagent A (20× Concentrate) | 2mL | 2-8℃ | | R777876E | DAB Chromogenic Reagent B (H₂O₂, 20× Concentrate) | 2mL | 2-8℃ | | R777876F | DAB Chromogenic Reagent C (TBS Buffer, 20× Concentrate) | 2mL | 2-8℃ |
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Reagents and Consumables to Be Prepared Separately:
1. Adhesive glass slides
2. PBS solution (pH 7.2–7.6)
3. EDTA Antigen Retrieval Solution or Citrate Buffer
Instructions for Use:
A. Staining Protocol for Paraffin Sections with Heat-Induced Antigen Retrieval
1. Prepare sections and perform routine deparaffinization and rehydration.
2. Incubate sections in 3% H₂O₂ at room temperature for 10 minutes to inactivate endogenous enzymes. Rinse with distilled water 3 times, 2 minutes each time.
3. Perform heat-induced antigen retrieval or enzymatic digestion as required based on antigen-antibody characteristics.
4. Add blocking solution and incubate at room temperature for 10 minutes. Decant excess liquid without rinsing.
5. Add appropriately diluted primary antibody (Rabbit IgG). Incubate at 20–37 °C for 1–2 hours, or overnight at 4 °C. Rinse with 0.01 M PBS 3 times, 2 minutes each time.
(Note: The dilution ratio and incubation conditions of the primary antibody directly affect staining intensity and background. If positive staining is weak, increase antibody concentration or prolong incubation time. If high background occurs, reduce antibody concentration or shorten incubation time.)
6. Add HRP-polymer conjugated Anti-Rabbit IgG, and incubate at 37 °C for 30 minutes. Rinse with PBS or TBS 3 times, 2 minutes each time.
7. DAB Chromogenic Reaction: Add 1 drop each of Reagent A, B and C to 1 mL of distilled water, mix well and apply onto sections. Develop color at room temperature under microscopic observation for 5–30 minutes. Rinse thoroughly with distilled water.
8. Counterstain lightly with hematoxylin, followed by dehydration, clearing, mounting and microscopic examination.
B. Staining Protocol for Blood Smears, Cell Smears and Frozen Sections
1. Pre-treat glass slides with Poly-L-Lysine for anti-fall effect. Prepare smears from anticoagulated blood after density gradient centrifugation; prepare smears or adherent monolayers with cultured cells. Air-dry frozen sections at room temperature with a fan.
2. Fix samples with 4% paraformaldehyde or acetone for 10–20 minutes.
3. Prepare working solution by mixing 1 part 30% H₂O₂ with 50 parts pure methanol. Immerse slides in the mixture at room temperature for 30 minutes to block endogenous peroxidase activity. Rinse with distilled water 1–2 times.
Follow Steps 3 to 8 of the paraffin section protocol for the remaining operations.
If unsatisfactory results are obtained from direct staining of frozen sections, perform heat-induced antigen retrieval referring to the paraffin section procedure, and follow Steps 3 to 8 accordingly.
Disclaimer & Safety Instructions:
1. This product is for research use only. It shall not be used for medical treatment, clinical diagnosis, food, cosmetics or other related applications.
2. Do not store this product in residential areas. Wear lab coat, disposable gloves and face mask during operation for personal safety and health.
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