Serial sections of resected tissue were immunostained for EMT mesenchymal biomarkers (S100A4, Vimentin), for the epithelial activation marker (epidermal growth factor receptor [EGFR]) and for vascularity using anti-Type-IV collagen for vessels in the Rbm. Sections of 3-µm thickness were mounted and stained with hematoxylin and eosin for morphological assessment and suitability for immunostaining. At room temperature, optimal sections were stained with the following antibodies: polyclonal anti-S100A4 (Dako, catalog no A5114, at 1:2,000 for 90 minutes), antivimentin monoclonal antibody (Dako, catalog no M7020, at 1:1,000 for 60 minutes), and Type-IV collagen monoclonal antibody (Dako, catalog no M0785, at 1:100 for 90 minutes) and monoclonal anti-EGFR (Dako, catalog no M3563, at 1:1,000 for 90 minutes). In each run, a section stained with immunoglobulin G1-negative control (X0931 clone DAKGO1; Dako Cytomation) was included to ensure absence of false-positive staining. Bound antibodies were elaborated by using horseradish peroxidase-conjugated DAKO Envision plus reagent (catalog no K4001, anti-mouse or K4003 anti-rabbit) and diaminobenzidine for a brown color resolution (catalog no K3468; Dako Cytomation). We have extensively used and published these methods.5 (link),6 (link),19 (link),20 (link)
Dako envision plus reagent
Manufactured by Agilent Technologies
DAKO Envision plus reagent is a visualization system used in immunohistochemistry and in situ hybridization procedures. It consists of a labeled polymer that is capable of binding to primary antibodies and detecting the target antigen or nucleic acid sequence.
Automatically generated - may contain errors
2 protocols using dako envision plus reagent
1
Immunohistochemical Assessment of EMT Markers
2
Immunohistochemistry of EMT Markers
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At room temperature, optimal sections were stained with the following antibodies: polyclonal anti-S100A4 (Dako cat no. A5114, 1:2000 for 90 minutes), anti-vimentin monoclonal antibody (Dako, cat no. M7020, 1:1000 for 60 minutes), N-cadherin (Abcam cat no. Ab98952, at 1:200 for 60 mins) and collagen IV monoclonal antibody (Dako, cat no. M0785, at 1:100 for 90 minutes) and monoclonal anti-EGFR (Dako cat no. M3563, at 1:1000 dilution for 90 minutes). In each run a section stained with immunoglobulin (Ig) G1-negative control (X0931 clone DAKGO1; Dako Cytomation) was included to ensure absence of false positive staining. Bound antibodies were elaborated by using horseradish peroxidase (HRP) conjugated DAKO Envision plus reagent (cat no. K4001, anti-mouse or K4003 anti-rabbit) and diaminobenzidine (DAB) for brown colour resolution (cat. no. K3468; Dako Cytomation). We have extensively used these methods [24] (link)[25] (link)[26] (link).
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