DEC-2005-IHC Envision+ method: Difference between revisions
From Labproto
(Daniela Elena Costea 2005, Oral Pathology, Haukeland Hospital, Bergen, Norway. PROTOCOL: IHC EnVision+ method for E-cadherin monoclonal antibody (R&D) on frozen sections of normal human oral mucosa (NHOM) and organotypic cultures of NHOM ~ 4 h) |
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[[Category:Protocols]] | |||
[[Category: Daniela Elena Costea]] | [[Category: Daniela Elena Costea]] | ||
[[Category: Oral Pathology Dept.]] | [[Category: Oral Pathology Dept.]] | ||
[[Category: Haukeland Hospital]] | [[Category: Haukeland Hospital]] | ||
[[Category: Norway]] | [[Category: Norway]] | ||
Latest revision as of 03:09, 30 August 2018
| Headline1 | Headline2 | Headline3 |
|---|---|---|
| Aceton cold 50% | In ventilation | 30 s |
| Aceton cold 100% | In ventilation | 5 min |
| Wash | TBS | 2 x 5 min |
| Primary antibody | E-cadherin in humidity chamber 1/9000 diluted in TBS |
60 min |
| Wash | TBS + TWEEN 20 TBS: 500ml of the stock (6.06 g TRIS/HCl from Merck L479219-136 + 1.39 g TRIS/base from Sigma T1503 in 1ldH2O check pH7.6) in 5l NaCl 0.87% (87g NaCl from Merck K33224404-418 in 10 l dH2O) |
2 x 5 min |
| Inactivation of peroxidase | peroxidase block from the EnVision+ kit | 5 min |
| Wash | TBS | 2 x 5 min |
| Secondary antibody | EnVision+ mouse (DAKO) |
30 min |
| Wash | TBS ± TWEEN 20 | 2 x 5 min |
| Visualisation | DAB 20 ul + 1 ml buffer (1 drop DAB+ 1 ml buffer) |
5 min |
| Wash | Running water | 10 min |
| Counterstain | Hematoxylin | 4 min |
| Wash | Running water | 10 min |
| Alchool | 70 % | 30 s |
| Alchool | 96 % | 2 min |
| Alchool | Absolut | 2 min |
| Toluen, xylol | In ventilation | 5 min |
| Mounting of sections | Eukit | 20 min |
