Mouse Immunohistochemistry Kit (HRP Polymer) is a high-sensitivity reagent kit. Using polymer labeling technology, horseradish peroxidase (HRP) is conjugated to the secondary antibody to form large-molecular antibody-enzyme polymers, which replace the secondary and tertiary antibodies used in conventional methods. These polymers deliver excellent signal amplification and strong tissue and cell penetration, making them ideal for immunohistochemical applications. Compared with the traditional three-step method, this series of detection reagents features simpler operation, faster assay, higher sensitivity and lower background staining.
Packing List:
| M777874 | Component | 1 Kit | Storage | | M777874A | Blocking Solution (5% BSA) | 6mL | 2-8℃ | | M777874B | Endogenous Peroxidase Blocker (3% H₂O₂) | 6mL | 2-8℃ | | M777874C | Anti-Mouse IgG (HRP-Polymer) | 6mL | 2-8℃ | | M777874D | DAB Chromogenic Reagent A (20× Concentrate) | 2mL | 2-8℃ | | M777874E | DAB Chromogenic Reagent B (H₂O₂, 20× Concentrate) | 2mL | 2-8℃ | | M777874F | 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. Antigen Retrieval and Staining Protocol for Paraffin Sections
1. Prepare sections and perform routine deparaffinization to hydration.
2. Endogenous Peroxidase Blocker (3% H₂O₂), incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with distilled water, 2 min × 3 times.
3. Perform heat-induced antigen retrieval or enzymatic digestion on sections if required, based on antigen-antibody properties.
4. Add Blocking Solution (5% BSA), incubate at room temperature for 10 min. Shake off excess liquid; do not wash.
5. Add appropriately diluted primary antibody (Mouse IgG), incubate at 20–37 °C for 1–2 h or overnight at 4 °C. Rinse with 0.01 M PBS, 2 min × 3 times. (The dilution, incubation time and temperature of the primary antibody directly affect staining intensity and background. In general, insufficient positive staining can be improved by increasing primary antibody concentration and extending incubation time; high background can be reduced by lowering primary antibody concentration and shortening incubation time.)
6. Add Anti-Mouse IgG (HRP-Polymer), incubate at 37 °C for 30 min. Rinse with PBS or TBS, 2 min × 3 times.
7. DAB color development: To 1 mL distilled water, add 1 drop each of DAB reagent A, DAB reagent B and DAB reagent C. Mix well and apply onto the section. Develop color at room temperature, monitor reaction time under microscope (typically 5–30 min). Wash with distilled water.
8. Light counterstain with hematoxylin, dehydrate, clear, mount slides and observe.
B. Staining Protocol for Blood Smears, Cell Preparations and Frozen Sections
1. Coat slides with adhesive agent (Poly-L-Lysine). Prepare smears from anticoagulated blood after density centrifugation; cultured cells can be smeared or grown on coverslips. Air-dry frozen sections under fan at room temperature.
2. Fix samples with 4% paraformaldehyde or acetone for 10–20 min.
3. Prepare blocking mixture: 1 part Endogenous Peroxidase Blocker (3% H₂O₂) mixed with 50 parts pure methanol. Immerse slides in the mixture at room temperature for 30 min to block endogenous peroxidase. Rinse with distilled water 1–2 times.
The remaining steps follow steps 3–8 of the paraffin section protocol.
If staining results of frozen sections are unsatisfactory, heat-induced antigen retrieval can be performed as described for paraffin sections, followed by steps 3–8 of the paraffin section protocol.
Precautions:
1. This product is for research use only. Do not use it for medicine, clinical diagnosis, therapy, food or cosmetic applications.
2. Do not store in residential areas. For your safety and health, wear a lab coat, disposable gloves and a mask during operation.
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