Immunohistochemical analysis of tumour samples stored as paraffin-embedded tissue was carried as previously described [42 (link)]. Laminin (1/100) and cleaved caspase 3 (1/100, #9664S, Cell Signaling, MA, USA) antibodies were used. Depending on the technique performed and the manufacturer’s specifications, different antibodies were used to detect myoepithelial cells: CD10 (1/100, #M7308, Dako, CA, USA), p63 (1/100, #sc-8431, Santa Cruz Biotechnologies, TE, USA) and αSMA (1/100) were used as primary antibodies. After washing the samples were incubated in the presence of HRP-conjugated secondary antibodies. After washing, samples were incubated with Vectastain ABC for 30 min in a humidified chamber followed by incubation in the presence of DAB substrate (FAST™ 3,3′-diaminobenzidine tablets, Sigma) for 5–10 min at RT, monitoring colour development by microscopy. Slides were washed with water and counterstained in 1:3 Gill II haematoxylin (Panreac) for 1 min. The slides were mounted with Cytoseal™ 60 (Thermo Scientific), left to dry and analysed by phase contrast microscopy.
Vectastain abc
Manufactured by Merck Group
Sourced in United States
Vectastain ABC is a laboratory kit used for immunohistochemistry and immunocytochemistry applications. The kit contains reagents required for the avidin-biotin complex (ABC) method, which is a widely used detection system for visualizing target antigens in tissue sections or cell samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using vectastain abc
1
Immunohistochemical Analysis of Tumor Samples
2
Quantifying NF-κB Expression in Pancreatic Tissue
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Pancreatic tissue sections were subjected to antigen retrieval with Retrievagen A
(Zymed Laboratories, Inc., San Francisco, CA, USA) and endogenous peroxidase
activity quenched with 3% H2O2 (Tianjin Jinqiang Chemical
Co. Ltd., Tianjin, China). Sections were then blocked in 2% BSA in phosphate
buffered saline (PBS) to prevent non-specific binding and incubated with primary
anti-NF-κB antibody (1:200; BD Pharmingen, Woburn, MA, USA) for 1 h at room
temperature. Sections were then washed with PBS and incubated with a
biotinylated rabbit anti-goat antibody (Thermo, Freemont, CA, USA) for 30 min at
37°C. Sections were developed using Vectastain ABC and 3,3′-diaminobenzidine
(Sigma-Aldrich, St. Louis, MO, USA). Sections were mounted and analyzed: Five
fields (magnification, 200×) were randomly selected from each section and the
average proportion of NF-κB-positive cells were counted using a true color,
multi-functional cell image analysis management system (Image-Pro Plus; Media
Cybernetics Inc., Rockville, MD, USA). All results were expressed as positive
units (pu).
(Zymed Laboratories, Inc., San Francisco, CA, USA) and endogenous peroxidase
activity quenched with 3% H2O2 (Tianjin Jinqiang Chemical
Co. Ltd., Tianjin, China). Sections were then blocked in 2% BSA in phosphate
buffered saline (PBS) to prevent non-specific binding and incubated with primary
anti-NF-κB antibody (1:200; BD Pharmingen, Woburn, MA, USA) for 1 h at room
temperature. Sections were then washed with PBS and incubated with a
biotinylated rabbit anti-goat antibody (Thermo, Freemont, CA, USA) for 30 min at
37°C. Sections were developed using Vectastain ABC and 3,3′-diaminobenzidine
(Sigma-Aldrich, St. Louis, MO, USA). Sections were mounted and analyzed: Five
fields (magnification, 200×) were randomly selected from each section and the
average proportion of NF-κB-positive cells were counted using a true color,
multi-functional cell image analysis management system (Image-Pro Plus; Media
Cybernetics Inc., Rockville, MD, USA). All results were expressed as positive
units (pu).
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