(HRP-Polymer)免疫组化试剂盒（Mouse）是高敏感性试剂盒。用聚合标记方法将过氧化物酶连接到二抗上，形成超大分子抗体-酶聚合物，替代传统方法中的二抗和三抗。这种聚合物拥有强大的信号放大能力，同时有很强的组织细胞渗透力，特别适合免疫组化的应用。此系列检测试剂与传统的三步法比较具有简单，快速，高敏感，低背景等特点。
包装清单：
M777874
| Component
| 1 Kit
| Storage
|
| M777874A | 封闭液（5%BSA）
| 6mL
| 2-8℃
|
| M777874B | 内源性过氧化物酶阻断剂（3%H2O2）
| 6mL
| 2-8℃
|
| M777874C | Anti-Mouse IgG（ HRP-Polymer）
| 6mL
| 2-8℃
|
| M777874D | DAB显色试剂A（DAB×20倍浓缩液）
| 2mL
| 2-8℃
|
| M777874E | DAB显色试剂B（H2O2×20倍浓缩液）
| 2mL
| 2-8℃
|
| M777874F | DAB显色试剂C（TBS×20倍浓缩缓冲液）
| 2mL
| 2-8℃
|
自备试剂及耗材：
1. 防脱玻片
2. PBS（pH7.2-7.6）
3. EDTA抗原修复液或枸橼酸盐缓冲液
使用说明：
A. 石蜡切片热修复抗原染色程序：1. 制备切片，常规脱蜡至水。
2. 内源性过氧化物酶阻断剂 (3%H₂O₂)，室温10 分钟以灭活内源性酶。蒸馏水洗2分钟×3次。
3. 根据抗原抗体情况，必要时对切片进行抗原热修复或消化处理。
4. 滴加封闭液 (5%BSA)，室温 10分钟。甩去多余液体，不洗。
5. 滴加适当稀释的一抗 (小鼠 IgG)，20-37℃ 1~2 小时或 4℃过夜。0.01M PBS 洗 2分钟×3次。（一抗的稀释度、孵育时间、温度与染色强度、背景有直接关系。一般来说，阳性染色强度不够时，可提高一抗浓度和延长孵育时间；背景过高时，可降低一抗浓度和缩短孵育时间。）
6. 滴加聚合HRP标记抗小鼠IgG， 37℃孵育 30 分钟，PBS 或 TBS 冲洗，2 分钟×3 次。
7. DAB显色：取1ml 蒸馏水，加入DAB显色试剂A、DAB显色试剂B、DAB显色试剂C各1滴，混匀后加至切片。室温显色，镜下控制反应时间，一般5-30 分钟。蒸馏水洗涤。
8. 苏木素轻度复染、脱水、透明、封片、观察。
B. 血涂片，细胞和冰冻切片染色程序：
1. 载玻片经防脱片剂处理 (Poly-L-Lysine)，抗凝血经分层离心后涂片；培养细胞也可涂片或贴片生长；冰冻切片室温风扇吹干。
2. 固定方案用4%多聚甲醛或丙酮固定10-20 分钟。
3. 内源性过氧化物酶阻断剂 (3%H₂O₂) 1份+纯甲醇50份混合，室温浸泡30 分钟，以灭活内源性过氧化物酶。蒸馏水洗1-2 次。
其余步骤和石蜡切片3-8 步相同。
如果冰冻切片直接染色结果不理想时，可以参照石蜡切片对切片进行热修复，步骤和石蜡切片3-8 步相同。
注意事项：
1. 本产品仅供科研使用，请勿用于医药、临床诊断或治疗，食品及化妆品等用途。
2. 请勿存放于普通住宅区，为了您的安全和健康，请穿好实验服并佩戴一次性手套和口罩操作。
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℃ |
|
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.
ZJ25F0724135
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