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지식나눔

지식나눔

rat 신장 조직에서 IHC 로 TNF와 IL-6를 보려고 합니다 DAB 발색좀 도와주세요

2012-06-18 org.kosen.entty.User@3b146c7c 오수진(jjioing)
  • 1
안녕하세요 초짜 석사생입니다 rat조직에서 IL-6,TNF를 IHC로 보려고 합니다 IHC 방법중에 DAB 발색을 하려면 DAB 용액을 만들어야하는데 어떻게 만들어야 할지 모르겠습니다. 처음 해보는 것이라 다 서툴러서 걱정이됩니다. DAB,0.05M Tris buffer(pH 7.2),3% H₂O₂외에 뭐가 더 필요한가요? 그리고 이 시약들을 얼만큼 섞어야하는지 모르겠습니다. 많은 고수님들 좀 알려주세요 IHC 전반적인 방법도 좀 알려주시면 정말 많은 도움이 될것 같습니다. 감사합니다 (^.^)(-.-)(^.^) 꼭좀 부탁드립니다 좀 급해서요....
  • IHC
  • DAB
  • rat
지식의 출발은 질문, 모든 지식의 완성은 답변! 
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    2012-06-18
    • 0
    DAB Peroxidase Substrate Solution Final Dilution: 0.05% DAB and 0.015% H2O2 in PBS, pH7.2(pH value is important! pH < 7.0 will reduce staining intensity. pH > 7.6 will cause background staining) * Stock Solutions: 1. 1% DAB (20x) in Distilled Water: Add 0.1g of DAB (3,3’-diaminobenzidine, Sigma, Cat#D8001 or DAB-tetrahydrocholoride) in 10 ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown color. Shake for 10 minutes and DAB should dissolve completely. Aliquot and store at ?20 °C. 2. 0.3% H2O2 (20x) in Distilled Water: Add 100ul of 30% H2O2 in 10 ml distilled water and mix well. Store at 4 °C or aliquot and store at ?20 °C. * Working Solution: 1. Add 5 drops of 1% DAB (1 drop = 50 ul) to 5 ml of PBS, pH7.2, and mix well. 2. Add 5 drops of 0.3% H2O2 and mix well. 3. Incubate sections for 1-3 minutes at room temperature. * Option: Add final conc. of 0.05% Nickel Ammonium Sulfate for enhancement staining (Stock sol.: 1% Nickel Ammonium Sulfate (20x) in Distilled Water: Add 0.1g of nickel ammonium sulfate in 10 ml distilled water. Shake to dissolve. Store at 4 °C or aliquot and store at ?20 °C.)
    답변수정
    DAB Peroxidase Substrate Solution Final Dilution: 0.05% DAB and 0.015% H2O2 in PBS, pH7.2(pH value is important! pH < 7.0 will reduce staining intensity. pH > 7.6 will cause background staining) * Stock Solutions: 1. 1% DAB (20x) in Distilled Water: Add 0.1g of DAB (3,3’-diaminobenzidine, Sigma, Cat#D8001 or DAB-tetrahydrocholoride) in 10 ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown color. Shake for 10 minutes and DAB should dissolve completely. Aliquot and store at ?20 °C. 2. 0.3% H2O2 (20x) in Distilled Water: Add 100ul of 30% H2O2 in 10 ml distilled water and mix well. Store at 4 °C or aliquot and store at ?20 °C. * Working Solution: 1. Add 5 drops of 1% DAB (1 drop = 50 ul) to 5 ml of PBS, pH7.2, and mix well. 2. Add 5 drops of 0.3% H2O2 and mix well. 3. Incubate sections for 1-3 minutes at room temperature. * Option: Add final conc. of 0.05% Nickel Ammonium Sulfate for enhancement staining (Stock sol.: 1% Nickel Ammonium Sulfate (20x) in Distilled Water: Add 0.1g of nickel ammonium sulfate in 10 ml distilled water. Shake to dissolve. Store at 4 °C or aliquot and store at ?20 °C.)
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