BCIP (5-Bromo-4-chloro-3-indolyl phosphate) 5-溴-4-氯-3-吲哚-磷酸盐＋NBT (四唑硝基蓝) 是碱性磷酸酶（AP）最佳的底物组合之一。在碱性磷酸酶的催化下，BCIP会被水解而产生强反应性的产物，该产物与NBT发生反应，形成不溶性的深蓝色至蓝紫色化合物。该试剂盒可用于AP系统的IHC 和Western Blot 实验的酶促显色。在AP催化下，在组织切片或印迹膜上结合了AP偶联物的地方产生深蓝色沉淀，可根据颜色反应来确定目的蛋白的位置及表达情况。
使用方法
1. AP显色工作液的配制
取20μL 150×NBT溶液加入到3mL碱性磷酸酶显色反应缓冲液，充分混合均匀，再加入10μL 300×BCIP溶液，配置成工作液，如需改变工作液体积，可按相同比例适量配制。工作液须现配现用，建议在1小时内使用完毕以保证最佳效果。
2. 样本预处理
对于检测干细胞等内源性碱性磷酸酯酶的组织或细胞样品，吸出培养基，PBS润洗3次，加入4%多聚甲醛固定1-2min。用TBST洗涤3-5次，每次3-5min。对于检测外源碱性磷酸酶的细胞样品、组织切片或膜，在与碱性磷酸酯酶标记的抗体或其它形式的探针孵育后，用TBST洗涤3-5次，每次3-5min。
3. 显色步骤
最后一次洗涤完毕去除洗涤液，加入适当体积上述显色工作液，室温避光孵育5-30min(可长达24h)，显色至预期深浅后，用蒸馏水清洗1-2次终止显色反应。如有必要可以用中性红染色液染色，显微镜下观察结果。
注意事项
工作液应现配现用，配制好的工作液1小时内有效。
工作液用量必需充足，保证完全覆盖组织片或印迹膜。
为获得最佳实验结果，请务必优化实验条件。
NBT有毒，使用时请采取必要的防护措施。
本产品仅用于科研，不能用于人体实验或人体治疗。
结果展示
图 1 HeLa 细胞碱性磷酸酶（BCIP/NBT）显色染色效果图
图 1. HeLa 细胞碱性磷酸酶（BCIP/NBT）显色染色效果图。 HeLa 细胞经 4% 多聚甲醛固定后，采用BCIP/NBT碱性磷酸酶显色试剂盒室温避光显色1h，光学显微镜下观察。蓝紫色沉淀为碱性磷酸酶活性阳性信号，示细胞内酶活性定位与表达水平。
BCIP (5-Bromo-4-chloro-3-indolyl phosphate) 5-bromo-4-chloro-3-indolyl-phosphate + NBT (tetrazolium nitro blue) is the best substrate for alkaline phosphatase (AP) One of the combination. Under the catalysis of alkaline phosphatase, BCIP will be hydrolyzed to produce a highly reactive product, which reacts with NBT to form an insoluble dark blue to blue-violet compound. This kit can be used for the enzymatic color development of IHC and Western Blot experiments of the AP system. Under AP catalysis, a dark blue precipitate is produced where AP conjugates are combined on tissue sections or blotting membranes. The location and expression of the target protein can be determined based on the color reaction.
Protocol
1. Preparation of AP Chromogenic Working Solution
Add 20 μL of 150× NBT solution to 3 mL of alkaline phosphatase chromogenic reaction buffer. Mix thoroughly. Then add 10 μL of 300× BCIP solution to prepare the working solution. If a different volume of working solution is required, adjust proportionally using the same ratio. The working solution must be prepared fresh immediately before use. It is recommended to use it within 1 hour to ensure optimal results.
2. Sample Pretreatment
For tissue or cell samples such as stem cells for detecting endogenous alkaline phosphatase: Aspirate the culture medium, rinse the samples 3 times with PBS, and fix with 4% paraformaldehyde for 1–2 minutes. Wash 3–5 times with TBST, each for 3–5 minutes. For cell samples, tissue sections, or membranes detecting exogenous alkaline phosphatase: After incubation with alkaline phosphatase-labeled antibodies or other forms of probes, wash 3–5 times with TBST, each for 3–5 minutes.
3. Chromogenic Development Step
Discard the washing solution after the final rinse, add an appropriate volume of the above chromogenic working solution, and incubate at room temperature in the dark for 5–30 minutes (incubation can be extended up to 24 hours). Once the desired color intensity is achieved, terminate the chromogenic reaction by rinsing 1–2 times with distilled water. Counterstain with Neutral Red staining solution if necessary, and observe the results under a microscope.
Notes
1. The working fluid should be prepared for immediate use, and the prepared working fluid will be effective within 1 hour.
2. The amount of working fluid must be sufficient to ensure complete coverage of the tissue sheet or blotting membrane.
3. In order to obtain the best experimental results, be sure to optimize the experimental conditions.
4. NBT is poisonous, please take necessary protective measures when using it.
5. This product is only used for scientific research, not for human experiments or human treatment.
Results Presentation
Figure 1 Alkaline phosphatase (BCIP/NBT) staining of HeLa cells
说明：图一请从附件中下载原图
Figure 1. Alkaline phosphatase (BCIP/NBT) staining of HeLa cells. HeLa cells were fixed with 4% paraformaldehyde, then stained using an alkaline phosphatase chromogenic kit with BCIP/NBT substrate system in the dark at room temperature for 1 hour. Images were captured under a light microscope. Blue–purple precipitates indicate positive signals of alkaline phosphatase activity, showing the localization and expression level of the enzyme in cells.
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