AR-V7 and AR-FL IHC was performed as previously described (30 (link), 31 (link)). Briefly, AR-V7 IHC was performed using recombinant rabbit monoclonal anti–AR-V7 antibody (Clone RM7, RevMAb Biosciences). Biopsies were first deparaffinized before antigen retrieval by microwaving (in Tris/EDTA buffer, pH 8.1) for 18 minutes at 800 W, and anti–AR-V7 antibody (1:500 dilution in Dako REAL diluent, Agilent Technologies) was incubated with tissue for 1 hour at room temperature. After washes, bound antibody was visualized using Dako EnVision Detection System (Agilent Technologies). Sections were counterstained with hematoxylin. Cell pellets from 22Rv1 (positive) and PC3 (negative) were used as controls. Rabbit IgGs were used as a further negative control. AR-FL IHC was performed using mouse monoclonal anti-AR antibody (AR441, Agilent Technologies). Biopsies were first deparaffinized before antigen retrieval using pH 8.1 Tris/EDTA solution heated in a water bath, and anti-AR antibody (1:1,000 dilution in Dako REAL diluent, Agilent Technologies) was incubated with tissue for 1 hour at room temperature. After washes, bound antibody was visualized using Dako EnVision Detection System (Agilent Technologies). Sections were counterstained with hematoxylin. Cell pellets from VCaP (positive) and PC3 (negative) were used as controls. Mouse IgGs were used as a further negative control.
Dako real diluent
Manufactured by Agilent Technologies
Sourced in Germany
Dako REAL diluent is a laboratory reagent used for the dilution of antibodies and other proteins in immunohistochemistry and immunocytochemistry applications. It is a buffered solution that helps maintain the stability and activity of the diluted biomolecules.
Automatically generated - may contain errors
2 protocols using dako real diluent
1
Immunohistochemical Detection of AR-V7 and AR-FL
2
Multiplex Immunohistochemistry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry [IHC] was performed on 2-µm sections of formalin-fixed, paraffin-embedded tissue [FFPE] according to a standard protocol using the ABC-AP Kit, Alkaline Phosphatase [Universal] [VECTASTAIN®, identifier: AK-5200, Vector labs, USA]. Briefly, after retrieval, sections were exposed to the primary antibody diluted in Dako REAL diluent [S202230-2, Agilent Technologies, Waldbronn, Germany] for 15 min at room temerature [RT], followed by incubation with the biotinylated secondary antibody [30 min, RT] and an incubation with the Vectastain ABC-AP reagent. After a TBS wash, a 5-min exposure to the Vector Blue alkaline phosphatase substrate was done and followed by Tris-buffered saline [TBS] wash. A second retrieval was performed, followed by another primary antibody incubation including all steps described above. The sections were exposed to the ImPACT Vector Red alkaline phosphatase substrate solution for 1-5 min, washed with deionized water and finally haematoxylin-counterstained. Dilutions and antigen-retrieval strategies for antibodies are listed in Supplementary Table S2 . Imaging was done using an Axio observer Z1, which was connected to an Axiocam 506 colour [both Carl Zeiss, Jena, Germany].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!