Immunocytochemistry from cell blocks was conducted as described elsewhere (Marwitz et al [18 (link)]. 2011) using mouse anti-E-Cadherin (1/400, Clone ECH6, Zytomed Systems, Germany), mouse anti TTF1 (1/100, Clone SPT24, Zytomed Systems, Germany), mouse anti-Podoplanin (1/100, Clone D2–40, Agilent Dako, Santa Clara, USA), rabbit anti-pro-SPC (1/100, polyclonal, ab90716, abcam, Cambridge, UK), mouse anti-Vimentin (1/1000, Clone V6, Zytomed Systems, Berlin, Germany) and polyclonal rabbit anti-Collagen I (1/1000, Abcam, Oxford, UK) diluted in antibody diluent (Zytomed Systems, Berlin, Germany). Negative controls were included under omission of primary antibody in every staining series.
Clone d2 40
Manufactured by Agilent Technologies
Sourced in Denmark
The Clone D2-40 is a monoclonal antibody product designed for use in immunohistochemical detection of the D2-40 antigen. The D2-40 antigen is expressed in lymphatic endothelial cells, making the Clone D2-40 antibody a useful tool for the identification of lymphatic vessels.
Automatically generated - may contain errors
Lab products found in correlation
Ultraview universal dab detection kit, by Roche (2 mentions)
Ventana benchmark xt, by Roche (2 mentions)
Antibody diluent, by Zytomed Systems (1 mentions)
Ab90716, by Abcam (1 mentions)
I view dab universal kit, by Roche (1 mentions)
Endogenous biotin blocking kit, by Roche (1 mentions)
Ventana benchmark ultra system, by Roche (1 mentions)
Benchmark xt, by Roche (1 mentions)
Clone clh2, by Leica Biosystems (1 mentions)
Clone clh5, by Leica Biosystems (1 mentions)
Clone mib 1, by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Dako omnis system, by Agilent Technologies (1 mentions)
Envision flex target retrieval solution high ph, by Agilent Technologies (1 mentions)
Clone e6h4, by Roche (1 mentions)
Clone ae1 ae3, by Agilent Technologies (1 mentions)
Ultraview universal alkaline phosphatase red detection kit, by Roche (1 mentions)
Clone jc70a, by Agilent Technologies (1 mentions)
Vector blocking kit, by Vector Laboratories (1 mentions)
6 protocols using clone d2 40
1
Immunocytochemistry Profiling of Cell Blocks
2
Immunohistochemical Analysis of Podoplanin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections of 4-µm thickness were immunostained with the AE1/3 antibody (PCK26, cocktail antibody, Ventana; Roche Tissue Diagnostics Japan; cat. no. 760-2595) and the podoplanin antibody (clone D2-40, Dako Agilent Technologies, Inc.; cat. no. M3619), respectively, using an automated immunostainer (VENTANA BenchMark ULTRA system; Ventana Medical Systems, Inc.) according to the manufacturers' protocol. The incubation with secondary antibodies and detection were carried out using the I–VIEW DAB Universal kit (Ventana Medical Systems, Inc.; cat. no. 760-041) and Endogenous Biotin Blocking kit (Ventana Medical Systems, Inc.; cat. no. 760-050).
Podoplanin expression in budding cells, which was confirmed by AE1/3 expression, was evaluated independently by the same authors mentioned above (HM and NK). The results were scored from 0 to 3 based on the intensity of the staining at the membrane or in the cytoplasm: 0, no reactivity; +1, weak; +2, moderate; and 3, marked, that is, overexpression (Fig. 2 ). For analysis, the results were divided into 2 groups: negative (score 0) and positive (from +1 to +3). The ly of the tumor tissue was evaluated using hematoxylin and eosin staining or podoplanin staining for detecting lymphatic vessels (negative; positive).
Podoplanin expression in budding cells, which was confirmed by AE1/3 expression, was evaluated independently by the same authors mentioned above (HM and NK). The results were scored from 0 to 3 based on the intensity of the staining at the membrane or in the cytoplasm: 0, no reactivity; +1, weak; +2, moderate; and 3, marked, that is, overexpression (
3
Immunohistochemical Characterization of Extrahepatic Bile Duct Carcinoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For histological examination, extrahepatic bile duct carcinoma specimens were routinely fixed with formalin, embedded in paraffin and thin-sectioned. Sections 4-μm-thick were mounted on saline-coated glass slides. Immunohistochemical examination was performed on deparaffinized sections using the standard avidin-biotin-peroxidase complex method with automated immunostainer (Benchmark XT; Ventana Medical System, Tucson, AZ, USA). The characteristic ‘inside-out’ pattern of IMPC was confirmed with the immunohistochemical MUC1 antibody. Furthermore, we investigated the phenotypes of IMPC using MUC1, MUC2, MUC5AC and MUC6 antibodies. Podoplanin (D2-40) was used for clarifying lymphatic invasion. The antibodies we used were: MUC1 (1:50, clone Ma696), MUC2 (1:50, clone Ccp), MUC5AC (1:100, clone CLH2), MUC6 (1:100, clone CLH5; all from Novocastra Laboratories), D2-40 (1:100, clone D2-40; Dako, Glostrup, Denmark).
4
Immunohistochemical Analysis of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FFPE sections were deparaffinized and rehydrated with a xylene and alcohol solution. Immunohistochemical staining was performed using a Ventana Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA) or a Dako Omnis System (Dako, Agilent Technologies, Carpinteria, CA, USA), according to the manufacturer's instructions. Antigen retrieval was performed using Cell Conditioning Solution (CC1; Ventana Medical Systems) or EnVision FLEX Target Retrieval Solution, High pH (Dako, Agilent Technologies). Sections were incubated with primary antibodies against D2-40 (1:100, clone D2-40, Dako), p16 (prediluted, clone E6H4, Ventana Medical Systems), and Ki-67 (1:150. clone MIB-1, Dako). After chromogenic visualization, using ultraView Universal DAB Detection Kit (Ventana Medical Systems) or EnVision FLEX /HRP (Dako, Agilent Technologies), slides were counterstained with hematoxylin. Appropriate positive and negative controls were concurrently stained to validate the staining method.
5
Immunohistochemical Analysis of Tumor Invasion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) slides searching for lymphovascular invasion images (cytokeratin plus podoplanin and cytokeratin plus CD31 dual color IHC slides) and tumor budding (cytokeratin IHC slides) were also analyzed. IHC were performed using the Ventana Benchmark XT® automated slide preparation system (Ventana- Roche Diagnostics, Meylan, France) using the UltraView Universal DAB Detection Kit (Ventana- Roche Diagnostics) and the UltraView Universal Alkaline Phosphatase Red Detection Kit (Ventana- Roche Diagnostics) with the following antibodies: for cytokeratin, clone AE1/AE3 (1:50 dilution, Dako, Glosstrup, Denmark), for podoplanin, clone D2–40 (pre-diluted, Dako) and for CD31, clone IC/70 A (1:20 dilution, Dako).
6
Immunohistochemical Evaluation of Endothelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed using the polymer detection method. After deparaffinization, antigen retrieval was performed in a pressure cooker after pretreatment with TRIS-EDTA at pH 9. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. Sections were then blocked for endogenous biotin with the Vector blocking kit. Slides were incubated overnight with anti-D2-40 antibody (1:100, clone D2-40; Dako, Glostrup, Denmark), anti-CD31 antibody (1:50, clone JC70A; Dako), and anti-factor 8 antibody (1:8000, polyclonal; Dako). Antigen-antibody complexes were detected using the Vector Avidin Biotin HRP, and visualization was performed with 3,3′-Diaminobenzidine and hydrogen peroxide. Hematoxylin was used for counterstaining for 3 minutes. Vessels of the tissue were used as (internal) positive control.
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!