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Immunohistochemistry of paraffin-embedded Human chronic tonsillitis using PD-L1/CD274/CD274 Rabbit mAb (A20344) at dilution of 1:200 (40x lens).Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Immunohistochemistry of paraffin-embedded Human intestinal adenocarcinoma (negative control samples) using PD-L1/CD274/CD274 Rabbit mAb (A20344) at dilution of 1:200 (40x lens).Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Immunohistochemistry of paraffin-embedded Human lung adenocarcinoma using PD-L1/CD274/CD274 Rabbit mAb (A20344) at dilution of 1:200 (40x lens).Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Immunohistochemistry of paraffin-embedded Human lung cancer sarcoma using PD-L1/CD274/CD274 Rabbit mAb (A20344) at dilution of 1:200 (40x lens).Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Immunohistochemistry of paraffin-embedded human placenta using PD-L1/CD274/CD274 Rabbit mAb (A20344) at dilution of 1:200 (40x lens).Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

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货号: A20344
促销价:   ¥1100
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产品内部验证

反应物种Human
产品应用测试种属及综合评分
IHCHuman:
推荐稀释比
  • IHC 1:50 - 1:200
理论分子量20kDa/33kDa
实际分子量Refer to figures
存储缓冲液Store at -20℃. Avoid freeze / thaw cycles.
Buffer: PBS with 0.02% thimerosal,0.05% BSA,50% glycerol,pH7.3.
阳性样本
细胞定位Cell membrane,Endomembrane system,Single-pass type I membrane protein
纯化方式Affinity purification

抗原信息

免疫原Recombinant protein of human PD‐L1 extracellular domain.
序列Email for sequence
进行序列比对

实验步骤

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实验步骤

免疫组织化学标准操作流程(石蜡切片——非磷酸化项目检测)

一、实验仪器及试剂

1、实验器材

微波炉、4℃冰箱、恒温恒湿箱、烘箱、光学显微镜、移液器、孵育湿盒、抗原修复盒。

2、实验试剂
  • (1) 缓冲液:0.01M PBS pH7.2±0.2; 0.01M pH7.2 PBST;
    试剂名称 1*PBS/L 试剂名称 1*PBST/L
    NaH2PO4 0.23g 1*PBS溶液 1L
    Na2HPO4 1.15g Tween 20 溶液 0.001L
    NaCl 8.5g 用稀HCl溶液和NaOH溶液调pH值
  • (2) 修复液(可选):
    pH6.0 0.01M柠檬酸修复液(1L):C6H8O7•H2O 0.4g;NaC6H5O7•2H2O 3g;pH6.0;
    pH7.0 0.01M PBS修复液(1L):KH2PO4 0.07g;NaCl 7.89g;Na2HPO4 0.45g;KCl 0.10g;pH7.2-7.4;
    pH8.0 0.05M Tris-EDTA修复液(1L):C4H11NO3 6.05g;C10H14N2Na2O8•2H2O 0.29g;用稀HCl调至pH8.0;
    pH9.0 0.01M Tris-EDTA修复液(1L):C4H11NO3 1.21g;C10H14N2Na2O8•2H2O 0.37g;pH9.0。
  • (3) 封闭液:5%空白山羊血清;
  • (4) 3%过氧化氢(需新鲜配制,30% H2O2与dH2O体积比1:9);
  • (5) 抗体稀释液:1X PBS / 5% 正常血清 / 0.3% Triton™ X-100;
  • (6) EnVision Systems检测体系, HRP显色系统: HRP RABBIT/MOUSE及配套DAB显色液;
  • (7) 脱蜡液、无水乙醇(分析纯)、去离子水(dH2O)、Mayer’s苏木素、中性树胶封片剂。

二、实验步骤

1.水化/脱蜡:
  • (1)烤片:将石蜡切片按同一朝向放置在切片架上,将其放入55℃的恒温箱中烤片30分钟;同时将脱蜡液1缸一起放入55℃的恒温箱中;
  • (2)脱蜡至水:将石蜡切片连同切片架一起放入脱蜡液1缸中,再一起从恒温箱中取出置于常温,5分钟后,将切片取出浸入到常温脱蜡液2缸中,并按照脱蜡液2、脱蜡液3、无水乙醇1、无水乙醇2、无水乙醇3的顺序依次将石蜡切片放入缸中,每缸5分钟;用流水清洗切片5分钟。
注意:流水清洗时水流不能直接对着切片;操作过程中需一直保持切片处于湿润状态。
2.内源性过氧化物酶灭活:
  • 将切片完全浸入到3%双氧水溶液中,室温,孵育10分钟;完成后流水清洗5分钟。
注意:内源性酶含量高的组织,可以延长灭活时间至15-20分钟;双氧水需新鲜配制,可提前5-10分钟配制3%的双氧水。
3.抗原修复(可选):
  • (1)方法一,微波热修复:将切片浸入盛有修复液的修复盒中,将抗原修复盒盖斜盖在修复盒上;整体放入微波炉中,高火加热3分钟后停火,微波炉内静置5分钟;再高火加热3分钟后停火,微波炉内静置5分钟;随后中低火加热1分钟后停火,微波炉内静置5分钟,然后将切片连同抗原修复盒拿出缓慢冷却至室温。待修复液温度降至室温后,用缓冲液PBS洗涤3次,每次1min。
  • (2)方法二,高压热修复:在高压锅中,加入抗原修复液,高火预热;待修复液沸腾后将切片置于其中,并完全浸泡组织,盖好锅盖,扣上压力阀,高火继续加热;待限压阀开始转动喷气后调至中火,同时开始计时2分钟;计时结束后离开热源,自然降压后将高压锅移入冷水中缓慢冷却。待修复液温度降至室温后,用缓冲液PBS洗涤3次,每次1min。
注意:修复液需完全浸没切片上组织;修复过程中严禁打开仪器或中断运行程序;修复完成后避免快速冷却;修复液可根据实验需求自行选用修复液。
4.染色:
  • (1)封闭:待修复液温度降至室温后,从修复盒中取出切片;用缓冲液PBS清洗切片2次,每次3分钟;去除切片上的缓冲液;在组织切片上滴加5%空白山羊血清封闭液;将切片水平放置在底部呈有水的孵育湿盒中,于37℃恒温孵育30分钟;
  • (2)一抗孵育:去除封闭液,在组织切片上滴加用缓冲液PBS稀释的一抗,水平放置于孵育湿盒中,于4℃孵育过夜;
  • (3)复温:将孵育湿盒取出室温复温15-30分钟,去除抗体工作液,用缓冲液PBST洗涤1次,5分钟;用缓冲液PBS洗涤3次,每次5分钟;
  • (4)二抗孵育:在组织切片上滴加二抗工作液后水平放置于孵育湿盒中,于37℃恒温孵育1h;
  • (5)去除切片上的溶液,用缓冲液PBST洗涤1次,5分钟;用缓冲液PBS洗涤3次,每次5分钟;
  • (6)显色:显色前5分钟,配置DAB显色工作液,C液:B液体积比1:100,充分混匀;在组织切片上滴加显色工作液,显微镜下密切观察颜色变化情况,通常10-60秒即可得到合适的染色强度;将切片浸入大量dH2O中即可终止显色;
  • (7)复染、返蓝:将切片浸入Mayer’s苏木素中复染切片,用流水清洗10分钟。
注意:染色过程中需一直保持切片处于湿润状态;染色过程中所有试剂使用过程中均需保证完全覆盖切片上组织。
5.脱水、封片:
  • (1)脱水:将清洗后的切片于无水乙醇中浸泡1次,10秒;高温(55℃-60℃)下完全干燥;
  • (2)封片:在切片中心滴加适量中性树胶,并加盖盖玻片。

靶点背景信息

This gene encodes an immune inhibitory receptor ligand that is expressed by hematopoietic and non-hematopoietic cells, such as T cells and B cells and various types of tumor cells. The encoded protein is a type I transmembrane protein that has immunoglobulin V-like and C-like domains. Interaction of this ligand with its receptor inhibits T-cell activation and cytokine production. During infection or inflammation of normal tissue, this interaction is important for preventing autoimmunity by maintaining homeostasis of the immune response. In tumor microenvironments, this interaction provides an immune escape for tumor cells through cytotoxic T-cell inactivation. Expression of this gene in tumor cells is considered to be prognostic in many types of human malignancies, including colon cancer and renal cell carcinoma. Alternative splicing results in multiple transcript variants.

基因ID29126
Swiss ProtQ9NZQ7
别名B7-H;B7H1;PDL1;PD-L1;PDCD1L1;PDCD1LG1;CD274
研究领域

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